High-affinity protein binders and uses thereof

Abstract

Provided herein are proteins that include a Vascular Endothelial Growth Factor A (VEGF-A) protein binding domain; proteins that include an Epstein-Barr Virus BCL-2 homolog (BHRF1) protein binding domain; proteins that include a Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) RBD protein binding domain; proteins that include an Interleukin-7 receptor subunit alpha (IL7R-α) protein binding domain; proteins that include a Programmed death-ligand 1 (PD-L1) protein binding domain; proteins that include a Tropomyosin-receptor kinase A (Trk-A) protein binding domain; and proteins that include an Interleukin-17 (IL-17A) protein binding domain.

Description

[0001] Atorney Docket No. 45288-0513WO1

[0002] HIGH-AFFINITY PROTEIN BINDERS AND USES THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 690, 167, filed on September 3, 2024, U.S. Provisional Patent Application No. 63 / 690,183, filed on September 3, 2024, U.S. Provisional Patent Application No. 63 / 690,188, filed on September 3, 2024, U.S. Provisional Patent Application No. 63 / 690,200, filed on September 3, 2024, U.S. Provisional Patent Application No. 63 / 690,214, filed on September 3, 2024, U.S. Provisional Patent Application No. 63 / 690,220, filed on September 3. 2024. and U.S. Provisional Patent Application No. 63 / 690,253, filed on September 3, 2024, which are incorporated herein by reference in their entireties.

[0005] FIELD

[0006] The present disclosure relates to the field of biotechnology, and more specifically, to high-affinity protein binders and uses thereof.

[0007] SEQUENCE LISTING

[0008] This application contains a Sequence Listing that has been submitted electronically as an XML file named 45288-0513WO1_SL_ST26. xml. The XML file, created on August 29, 2025, is 34,493 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0009] BACKGROUND

[0010] Protein-protein interaction is a fundamental aspect of protein function, and proteinbinding proteins are a basic building block for therapeutics, diagnostics, and biomedical research. Traditionally, antibodies, nanobodies, and other scaffolds such as DARPins or affibodies are developed into binders against a wide range of targets by immunization or directed evolution. However, experimental selection does not afford control over the target epitope and is often too laborious for routine research applications. Computational design of binders de novo, without using a natural protein as a starting point, can target pre-specified epitopes and generate binders that are smaller, more thermostable, and easier to express than antibodies. Atorney Docket No. 45288-0513WO1

[0011] Recently, deep-learning based models have achieved major advances in biomolecular structure prediction and protein design. This has enabled progress on key scientific and societal challenges, including the prediction and design of protein-protein interactions. It is now possible to obtain computationally designed protein binders to some targets without initial high-throughput screening. High binding affinity without preliminary' experimental optimization has also been achieved in some cases, such as for small peptides or disordered targets. However, success rates remain low against convex or polar epitopes, the affinity of the initial designs is usually poor, and many targets remain intractable.

[0012] SUMMARY

[0013] Some embodiments provide a protein comprising a Vascular Endothelial Grow th Factor A (VEGF-A) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0014] Some embodiments provide a nucleic acid comprising a sequence encoding any of the proteins described herein (e.g., SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5).

[0015] Some embodiments provide a vector comprising the nucleic acid comprising a sequence encoding any of the proteins described herein (e.g., SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5).

[0016] Some embodiments provide a cell comprising the nucleic acid or the vector described herein.

[0017] Some embodiments provide a pharmaceutical composition comprising any one of the proteins described herein (e g., SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5), the nucleic acid, or the vector, or the cell described herein; and a pharmaceutically acceptable carrier.

[0018] Some embodiments provide a method of treating a VEGF-A associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a protein described herein (e.g., SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5), or a pharmaceutical composition comprising the same.

[0019] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 2, SEQ ID NO: 3. SEQ ID NO: 4, or SEQ ID NO: 5). or a pharmaceutical composition comprising the same, for use in therapy. Atorney Docket No. 45288-0513WO1

[0020] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5). or a pharmaceutical composition comprising the same, for use in a method of treating a VEGF-A associated disease in a subject in need thereof.

[0021] Some embodiments provide a protein comprising an Epstein-Barr Virus BCL-2 homolog (BHRF1) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0022] Some embodiments provide a nucleic acid comprising a sequence encoding a protein as described herein (e.g., SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9).

[0023] Some embodiments provide a vector comprising the nucleic acid comprising a sequence encoding a protein as described herein (e.g., SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9).

[0024] Some embodiments provide a cell comprising the nucleic acid or the vector as described herein.

[0025] Some embodiments provide a pharmaceutical composition comprising a protein described herein (e.g., SEQ ID NO: 7. SEQ ID NO: 8, or SEQ ID NO: 9), or the nucleic acid, or the vector, or the cell, described herein; and a pharmaceutically acceptable carrier.

[0026] Some embodiments provide a method of treating a BHRF1 associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a protein described herein (e.g., SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9). or a pharmaceutical composition comprising the same.

[0027] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9), or a pharmaceutical composition comprising the same, for use in therapy.

[0028] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9), or a pharmaceutical composition comprising the same, for use in a method of treating a BHRF1 associated disease in a subject in need thereof.

[0029] Some embodiments provide a protein comprising a Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) RBD protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0030] Some embodiments provide a nucleic acid comprising a sequence encoding a protein described herein (e.g., SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16). Atorney Docket No. 45288-0513WO1

[0031] Some embodiments provide a vector comprising a nucleic acid described herein (e.g., encoding a protein described herein, such as SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16).

[0032] Some embodiments provide a cell comprising a nucleic acid or a vector as described herein.

[0033] Some embodiments provide a pharmaceutical composition comprising a protein described herein (e.g.. SEQ ID NO: 11. SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14. SEQ ID NO: 15, or SEQ ID NO: 16), or a nucleic acid, or a vector, or a cell, as described herein; and a pharmaceutically acceptable carrier.

[0034] Some embodiments provide a method of treating a SARS-CoV-2 associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a protein described herein (e.g., SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16), or a pharmaceutical composition comprising the same.

[0035] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15. or SEQ ID NO: 16). or a pharmaceutical composition comprising the same, for use in therapy.

[0036] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16). or a pharmaceutical composition comprising the same, for use in a method of treating a SARS- CoV-2 associated disease in a subject in need thereof.

[0037] Some embodiments provide a protein comprising an Interleukin-7 receptor subunit alpha (IL7R-a) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

[0038] Some embodiments provide a nucleic acid comprising a sequence encoding a protein as described herein (e.g., SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20).

[0039] Some embodiments provide a vector comprising the nucleic acid comprising a sequence encoding a protein as described herein (e.g., SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20).

[0040] Some embodiments provide a cell comprising the nucleic acid or the vector as described herein.

[0041] Some embodiments provide a pharmaceutical composition comprising a protein described herein (e.g., SEQ ID NO: 18. SEQ ID NO: 19, or SEQ ID NO: 20), or the nucleic acid, or the vector, or the cell, described herein; and a pharmaceutically acceptable carrier. Atorney Docket No. 45288-0513WO1

[0042] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20), or a pharmaceutical composition comprising the same for use in therapy.

[0043] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20), or a pharmaceutical composition comprising the same for use in a method of treating an IL7R-A associated disease in a subject in need thereof.

[0044] Some embodiments provide a method of treating an IL7R-A associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a protein described herein (e.g., SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20), or a pharmaceutical composition comprising the same.

[0045] Some embodiments provide a protein comprising a Programmed death-ligand 1 (PD- Ll) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0046] Some embodiments provide a nucleic acid comprising a sequence encoding a protein as described herein (e.g.. SEQ ID NO: 22. SEQ ID NO: 23, or SEQ ID NO: 24).

[0047] Some embodiments provide a vector comprising the nucleic acid comprising a sequence encoding a protein as described herein (e g., SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24).

[0048] Some embodiments provide a cell comprising the nucleic acid or the vector as described herein.

[0049] Some embodiments provide a pharmaceutical composition comprising a protein described herein (e.g., SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24), or the nucleic acid, or the vector, or the cell, described herein; and a pharmaceutically acceptable carrier.

[0050] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24), or a pharmaceutical composition comprising the same for use in therapy.

[0051] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24), or a pharmaceutical composition comprising the same for use in a method of treating a PD-L1 associated disease in a subject in need thereof.

[0052] Some embodiments provide a method of treating a PD-L1 associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a protein described herein (e.g., SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24), or a pharmaceutical composition comprising the same. Atorney Docket No. 45288-0513WO1

[0053] Some embodiments provide a protein comprising a Tropomyosin-receptor kinase A (Trk-A) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

[0054] Some embodiments provide a nucleic acid comprising a sequence encoding a protein as described herein (e.g., SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28).

[0055] Some embodiments provide a vector comprising the nucleic acid comprising a sequence encoding a protein as described herein (e.g.. SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28).

[0056] Some embodiments provide a cell comprising the nucleic acid or the vector as described herein.

[0057] Some embodiments provide a pharmaceutical composition comprising a protein described herein (e.g., SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28), or the nucleic acid, or the vector, or the cell, described herein; and a pharmaceutically acceptable carrier.

[0058] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28), or a pharmaceutical composition comprising the same for use in therapy.

[0059] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28), or a pharmaceutical composition comprising the same for use in a method of treating a Trk-A associated disease in a subject in need thereof.

[0060] Some embodiments provide a method of treating a Trk-A associated disease in a subj ect in need thereof, the method comprising administering to the subject a therapeutically effective amount of a protein described herein (e.g., SEQ ID NO: 26, SEQ ID NO: l, or SEQ ID NO: 28), or a pharmaceutical composition comprising the same.

[0061] Some embodiments provide a protein comprising an Interleukin- 17 (IL-17A) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

[0062] Some embodiments provide a nucleic acid comprising a sequence encoding a protein as described herein (e.g.. SEQ ID NO: 30. SEQ ID NO: 31, or SEQ ID NO: 32).

[0063] Some embodiments provide a vector comprising the nucleic acid comprising a sequence encoding a protein as described herein (e.g., SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32).

[0064] Some embodiments provide a cell comprising the nucleic acid or the vector as described herein. Atorney Docket No. 45288-0513WO1

[0065] Some embodiments provide a pharmaceutical composition comprising a protein described herein (e.g., SEQ ID NO: 30. SEQ ID NO: 31, or SEQ ID NO: 32), or the nucleic acid, or the vector, or the cell, described herein; and a pharmaceutically acceptable carrier.

[0066] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32), or a pharmaceutical composition comprising the same for use in therapy.

[0067] Some embodiments provide a protein described herein (e.g., SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32), or a pharmaceutical composition comprising the same for use in a method of treating an IL-17A associated disease in a subject in need thereof.

[0068] Some embodiments provide a method of treating an IL-17A associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a protein described herein (e.g., SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32), or a pharmaceutical composition comprising the same.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0070] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0071] BRIEF DESCRIPTION OF DRAWINGS

[0072] FIG. 1 shows a crystal structure (light yellow) and hotspot residues (dark yellow spheres) of the VEGF-A target protein for binder design experiments described herein. VEGF- A is a disulfide-linked homodimer.

[0073] FIGs. 2A-2D show biochemical characterization of a representative binder for VEGF- A. FIG. 2A shows a design model, FIG. 2B shows HTRF equilibrium saturation binding and Atorney Docket No. 45288-0513WO1

[0074] KDS fited from the 1: 1 binding model, FIG. 2C shows yeast display on interface mutants and competitive inhibition, and FIG. 2D shows circular dichroism spectra at 20°C and 95°C of the representative binder for VEGF-A.

[0075] FIGs. 3A-3B show inhibition of VEGF signaling by designed binders. FIG. 3A shows Western blots of phosphorylated ERK and AKT from HUVEC cells after 2 to 60 minutes of treatment with designed binders GDM VEGFA 54 or GDM VEGFA 71 and VEGF-A stimulation. Inhibition of VEGF-A signaling is observed by a reduction in pERK and pAKT band intensity relative to VEGF-A-only ("no inhibitor") control. Inhibition by the binders is comparable to existing small-molecule VEGFR2 inhibitor ki8751 and VEGF-A clinical mAb Bevacizumab. FIG. 3B shows a schematic representation of the VEGF-A signaling pathway. VEGF-A binding leads to dimerization of VEGFR, phosphorylation of VEGFR and downstream signaling cascade leading to ERK and AKT phosphorylation.

[0076] FIGs. 4A-4D show experimental structures of binders to VEGF-A. FIG. 4A shows crystal structure of complex between VEGF-A homodimer (yellow) and design GDM VEGFA 71 (blue), aligned to AF3 prediction (grey) on VEGF-A (binder Ca RMSD = 1.65 A). FIG. 4B shows rotated view of binder monomer (binder-aligned binder Ca RMSD = 0.78 A. FIG. 4C shows packing of hydrophobic sidechains of the binder at the interface. Most have near-perfect agreement between design and structure, except Vall7, Ilel9, and Ile81, which have slight deviations. FIG. 4D shows a designed hydrogen bond betw een His24 of the binder and Tyr25 of VEGF-A.

[0077] FIGs. 5A-5B show circular dichroism spectra and thermal melts of the identified VEGF-A binders.

[0078] FIGs. 6A-6B show equilibrium saturation binding by homogeneous time-resolved fluorescence (HTRF). HTRF equilibrium saturation binding data for all designs that expressed and displayed observable binding. Control binders from the literature are shown in black. The binder design was held at a fixed concentration of either 0. 1 nM or 1 nM and the target protein concentration w as titrated. Parentheses indicate the fitted KD value from a generalized (squareroot form) 1 : 1 binding equation and the number of replicates for each design.

[0079] FIG. 7 shows a structure showing binder interface and mutations made to knock-out the interaction to verily it is binding as intended by the design model. Mutation at the binding interface shown in red, and other interface positions colored in light green. All non-binding regions of the binder design are colored in yellow.

[0080] FIGs. 8A-8B shows interface mutation and competition experiments on the identified

[0081] VEGF-A binders. Atorney Docket No. 45288-0513WO1

[0082] FIG. 9 shows HTRF binding signal (background subtracted) of a subset of top binders against each of the target proteins.

[0083] FIG. 10 shows functional validation of VEGF-A binders in HUVEC cells. Western blots of phosphorylated and total ERK, AKT, and VEGFR2 after treatment with VEGF-A binders, known VEGF-A or VEGFR2 inhibitors, or no inhibitor, demonstrating that the identified VEGF-A binders exhibit inhibition of VEGF-A signaling via VEGFR2 in HUVEC cells.

[0084] FIG. 11 shows a crystal structure (light yellow) and hotspot residues (dark yellow spheres) of the BHRF1 target protein for binder design experiments described herein.

[0085] FIGs. 12A-12G show' biochemical characterization of a representative binder for BHRF1.

[0086] FIGs. 13A-13B show equilibrium saturation binding by homogeneous time-resolved fluorescence (HTRF). HTRF equilibrium saturation binding data for all designs that expressed and displayed observable binding. Control binders from the literature are shown in black. The binder design was held at a fixed concentration of either 0. 1 nM or 1 nM and the target protein concentration was titrated. Parentheses indicate the fitted KD value from a generalized (squareroot form) 1: 1 binding equation and the number of replicates for each design.

[0087] FIG. 14 show s a structure showing binder interface and mutations made to knock-out the interaction to verify it is binding as intended by the design model. Mutation at the binding interface shown in red, and other interface positions colored in light green. All non-binding regions of the binder design are colored in yellow.

[0088] FIG. 15A-15B show s interface mutation and competition experiments on the identified BHRF1 binders.

[0089] FIG. 16 shows a crystal structure (light yellow) and hotspot residues (dark yellow spheres) of the SARS_CoV-2 RBD target protein for binder design experiments described herein.

[0090] FIGs. 17A-17F show biochemical characterization of a representative binder for SARS_CoV-2 RBD. FIG. 17A shows a design model, FIG. 17B shows HTRF equilibrium saturation binding and KDS fitted from the 1: 1 binding model. FIGs. 17C and 17E show yeast display on interface mutants and competitive inhibition, and FIGs. 17D and 17F show s circular dichroism spectra at 20°C and 95°C of the representative binder for SARS_CoV-2 RBD.

[0091] FIG. 18 shows inhibition of SARS-CoV-2 viral infection by designed binders. Results show 50% inhibitory concentration (EC50) of 4 designed SC2RBD binders in a virus Atorney Docket No. 45288-0513WO1 neutralization assay against 4 SARS-CoV-2 variants. Plotted are means and 95% confidence intervals from fits to 2 biological replicates.

[0092] FIG. 19 shows experimental structures of binders to SARS-CoV-2 spike. Results show cryo-EM structures of designed binders (blue) in complex with SARS-CoV-2 spike protein (y ellow), aligned to AF3 prediction (grey) on spike protein. Values are shown for the cryo-EM structure resolution and target-aligned binder Ca RMSDs between AF3-predicted and experimental structures.

[0093] FIGs. 20A-20C show circular dichroism spectra and thermal melts of the identified SARS_CoV-2 RBD binders.

[0094] FIGs. 21A-21B show equilibrium saturation binding by homogeneous time-resolved fluorescence (HTRF). HTRF equilibrium saturation binding data for all designs that expressed and displayed observable binding. Control binders from the literature are shown in black. The binder design was held at a fixed concentration of either 0. 1 nM or 1 nM and the target protein concentration w as titrated. Parentheses indicate the fitted KD value from a generalized (squareroot form) 1 : 1 binding equation and the number of replicates for each design.

[0095] FIG. 22 shows structural locations of interface mutations.

[0096] FIGs. 23A-23C show interface mutation and competition experiments on the identified SARS_CoV-2 RBD binders.

[0097] FIG. 24 shows SARS-CoV-2 neutralization assay for four selected binders over four variants of interest. SARS-CoV-2 virus neutralization assay was performed in Vero cells by the Francis Crick Covid Surveillance Unit. Each plot consists of 160 independent data points (4 technical replicates, 2 biological replicates, 40 independent titrations). EC50 values were calculated using nonlinear regression with a 4-parameter dose response curve fit. Fits are shown only when standard error on EC50 was within one order of magnitude and the percentage infected is reduced to at least 60%. 95% confidence intervals are shown as shaded areas. All four binders tested successfully neutralize the England2 / Ancestral variant of the SARS-CoV-2 virus.

[0098] FIGs. 25A-25E show SARS-CoV-2 cryo-EM data processing. FIG. 25A shows 2D class averages of particle images from the SC2RBD 129 dataset corresponding to dissociated spikes; the scale bar is 10 nm. FIG. 25B shows the result of 3D classification of the same particles into 5 classes; the number of particles belonging to each class is indicated underneath. The two best 3D classes collectively comprising 206337 particles used for the final 3D reconstruction are boxed. FIG. 25C shows half-map Fourier shell correlation (FSC) for each of the final reconstructions. Dotted line indicates the gold-standard cut-off at FSC of 0.143. Atorney Docket No. 45288-0513WO1

[0099] FIG. 25D shows final reconstruction of SI in complex with GDM_SC2RBD_129 ligand in two orthogonal orientations. The cryo-EM map is shown as a transparent grey surface with docked SI (from PDB ID 7ZBU, blue) and the ligand (orange) chains as cartoons. Locations of the ligand, individual SI domains (RBD, NTD, SD1 and SD2) as well as select glycans (attached to Asn residues 165, 282, and 343) are indicated. FIG. 25E shows SARS-CoV-2 receptor binding domain (yellow) bound to the binder design (blue) was fitted together into the CryoEM density map (transparent grey surface).

[0100] FIG. 26 shows a crystal structure (light yellow) and hotspot residues (dark yellow spheres) of the IL7R-a target protein for binder design experiments described herein.

[0101] FIGs. 27A-27D show biochemical characterization of a representative binder for IL7R- a. FIG. 27A shows a design model, FIG. 27B shows HTRF equilibrium saturation binding and KDS fitted from the 1 : 1 binding model, FIG. 27C shows yeast display on interface mutants and competitive inhibition, and FIG. 27D shows circular dichroism spectra at 20°C and 95°C of the representative binder for IL7R-a.

[0102] FIGs. 28A-28B show circular dichroism spectra and thermal melts of the identified IL7R-a binders.

[0103] FIGs. 29A-29B show equilibrium saturation binding by homogeneous time-resolved fluorescence (HTRF). HTRF equilibrium saturation binding data for all designs that expressed and displayed observable binding. Control binders from the literature are shown in black. The binder design was held at a fixed concentration of either 0. 1 nM or 1 nM and the target protein concentration was titrated. Parentheses indicate the fitted KD value from a generalized (squareroot form) 1: 1 binding equation and the number of replicates for each design.

[0104] FIG. 30 shows a structure showing binder interface and mutations made to knock-out the interaction to verily it is binding as intended by the design model. Mutation at the binding interface shown in red, and other interface positions colored in light green. All non-binding regions of the binder design are colored in yellow.

[0105] FIGs. 31A-31C show interface mutation and competition experiments on the identified IL7R-a binders.

[0106] FIG. 32 shows a crystal structure (light yellow) and hotspot residues (dark yellow spheres) of the PD-L1 target protein for binder design experiments described herein.

[0107] FIGs. 33A-33D show biochemical characterization of a representative binder for PD- Ll. FIG. 33A shows a design model, FIG. 33B shows HTRF equilibrium saturation binding and KDS fitted from the 1 : 1 binding model, FIG. 33C shows yeast display on interface mutants Atorney Docket No. 45288-0513WO1 and competitive inhibition, and FIG. 33D shows circular dichroism spectra at 20°C and 95°C of the representative binder for PD-L1.

[0108] FIGs. 34A-34B show circular dichroism spectra and thermal melts of the identified PD-L1 binders.

[0109] FIGs. 35A-35B show equilibrium saturation binding by homogeneous time-resolved fluorescence (HTRF). HTRF equilibrium saturation binding data for all designs that expressed and displayed observable binding. Control binders from the literature are shown in black. The binder design was held at a fixed concentration of either 0. 1 nM or 1 nM and the target protein concentration was titrated. Parentheses indicate the fitted KD value from a generalized (squareroot form) 1 : 1 binding equation and the number of replicates for each design.

[0110] FIG. 36 shows a structure showing binder interface and mutations made to knock-out the interaction to verily it is binding as intended by the design model. Mutation at the binding interface shown in red, and other interface positions colored in light green. All non-binding regions of the binder design are colored in yellow .

[0111] FIG. 37 shows interface mutation and competition experiments on the identified PD- L1 binder.

[0112] FIG. 38 shows a crystal structure (light yellow) and hotspot residues (dark yellow spheres) of the Trk-A target protein for binder design experiments described herein.

[0113] FIGs. 39A-39D show biochemical characterization of a representative binder for Trk- A. FIG. 39A shows a design model, FIG. 39B shows HTRF equilibrium saturation binding and KDS fitted from the 1 : 1 binding model, FIG. 39C shows yeast display on interface mutants and competitive inhibition, and FIG. 39D shows circular dichroism spectra at 20°C and 95 °C of the representative binder for Trk-A.

[0114] FIGs. 40A-40B show circular dichroism spectra and thermal melts of the identified Trk-A binders.

[0115] FIG. 41 shows equilibrium saturation binding by homogeneous time-resolved fluorescence (HTRF). HTRF equilibrium saturation binding data for all designs that expressed and displayed observable binding. Control binders from the literature are shown in black. The binder design was held at a fixed concentration of either 0. 1 nM or 1 nM and the target protein concentration was titrated. Parentheses indicate the fitted KD value from a generalized (squareroot form) 1:1 binding equation and the number of replicates for each design.

[0116] FIG. 42 shows a structure showing binder interface and mutations made to knock-out the interaction to verily it is binding as intended by the design model. Mutation at the binding Atorney Docket No. 45288-0513WO1 interface shown in red, and other interface positions colored in light green. All non-binding regions of the binder design are colored in yellow.

[0117] FIGs. 43A-43B show interface mutation and competition experiments on the identified Trk-A binders.

[0118] FIG. 44 shows a crystal structure (light yellow) and hotspot residues (dark yellowspheres) of the IL-17A target protein for binder design experiments described herein.

[0119] FIGs. 45A-45D show biochemical characterization of a representative binder for ILIYA. FIG. 45A shows a design model, FIG. 45B shows HTRF equilibrium saturation binding and KDS fitted from the 1 : 1 binding model, FIG. 45C shows yeast display on interface mutants and competitive inhibition, and FIG. 45D shows circular dichroism spectra at 20°C and 95°C of the representative binder for IL- 17 A.

[0120] FIGs. 46A-46B show circular dichroism spectra and thermal melts of the identified IL- 17A binders.

[0121] FIG. 47 shows equilibrium saturation binding by homogeneous time-resolved fluorescence (HTRF). HTRF equilibrium saturation binding data for all designs that expressed and displayed observable binding. Control binders from the literature are shown in black. The binder design was held at a fixed concentration of either 0. 1 nM or 1 nM and the target protein concentration was titrated. Parentheses indicate the fitted KD value from a generalized (squareroot form) 1 : 1 binding equation and the number of replicates for each design.

[0122] FIG. 48 shows a structure showing binder interface and mutations made to knock-out the interaction to verily it is binding as intended by the design model. Mutation at the binding interface shown in red, and other interface positions colored in light green. All non-binding regions of the binder design are colored in yellow.

[0123] FIGs. 49A-49C show interface mutation and competition experiments on the identified IL-17A binders.

[0124] DETAILED DESCRIPTION

[0125] Computational design of protein-binding proteins is a fundamental capability with broad utility in biomedical research and biotechnology. Recent methods have made strides against some target proteins, but on-demand creation of high-affinity binders without multiple rounds of experimental testing remains an unsolved challenge.

[0126] A family of machine learning models (“AlphaProteo”) can be used for protein design, generating binders with 3- to 300-fold better binding affinities and higher experimental success Atorney Docket No. 45288-0513WO1 rates than the best existing methods. Binding proteins against seven different target proteins (e.g., BHRF1, SARS-CoV-2 spike protein receptor-binding domain (SC2RBD), Interleukin-7 Receptor-a (IL-7RA), Programmed Death-Ligand 1 (PD-L1), Tropomyosin Receptor Kinase A (TrkA), Interleukin-17A (IL-17A), and Vascular Endothelial Growth Factor A (VEGF-A)) were designed.

[0127] Provided herein are proteins that include Vascular Endothelial Growth Factor A (VEGF-A) protein binding domain, nucleic acids encoding the same, cells including any of these nucleic acids or proteins, compositions including any of these proteins, nucleic acids, and cells, and methods of treating a subject having a VEGF-A associated disease using any of the compositions described herein.

[0128] Provided herein are proteins that include an Epstein-Barr Virus BCL-2 homolog (BHRF1) protein binding domain, nucleic acids encoding the same, cells including any of these nucleic acids or proteins, compositions including any of these proteins, nucleic acids, and cells, and methods of treating a subject having a BHRF1 associated disease using any of the compositions described herein.

[0129] Provided herein are proteins that include a Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) RBD protein binding domain, nucleic acids encoding the same, cells including any of these nucleic acids or proteins, compositions including any of these proteins, nucleic acids, and cells, and methods of treating a subject having a SARS-CoV-2 associated disease using any of the compositions described herein.

[0130] Provided herein are proteins that include an Interleukin-7 receptor subunit alpha (IL7R- a) protein binding domain, nucleic acids encoding the same, cells including any of these nucleic acids or proteins, compositions including any of these proteins, nucleic acids, and cells, and methods of treating a subject having an IL7R-a associated disease using any of the compositions described herein.

[0131] Provided herein are proteins that include a Programmed death-ligand 1 (PD-L1) protein binding domain, nucleic acids encoding the same, cells including any of these nucleic acids or proteins, compositions including any of these proteins, nucleic acids, and cells, and methods oftreating a subject having a PD-L1 associated disease using any of the compositions described herein.

[0132] Provided herein are proteins that include a Tropomyosin receptor kinase A (Trk-A) protein binding domain, nucleic acids encoding the same, cells including any of these nucleic acids or proteins, compositions including any of these proteins, nucleic acids, and cells, and Atorney Docket No. 45288-0513WO1 methods of treating a subject having a Trk-A associated disease using any of the compositions described herein.

[0133] Provided herein are proteins that include an Interleukin- 17 (IL-17A) protein binding domain, nucleic acids encoding the same, cells including any of these nucleic acids or proteins, compositions including any of these proteins, nucleic acids, and cells, and methods of treating a subject having an IL-17A associated disease using any of the compositions described herein.

[0134] Various non-limiting aspects of these proteins, nucleic acids, cells, compositions, and methods are described herein, and can be used in any combination without limitation. Additional aspects of various components of methods of making and using a protein that includes a VEGF-A protein binding domain are known in the art.

[0135] As used herein, the term “about’; when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about’’ in that context. For example, in some embodiments, the term “about’’ may encompass a range of values that are within 10% of the indicated value, for example 10%, 9%, 8%, 7%, 6%, 5%, 4%. 3%, 2%. 1%. or less of the referred value.

[0136] As used herein, the term “binding” typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell).

[0137] As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. A binding agent that interacts with one particular target when other potential targets are present is said to “bind specifically ” to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations. Atorney Docket No. 45288-0513WO1

[0138] Reference is made herein to certain specific binding targets, including isoforms, fragments, and variants thereof As used herein, a 'fragment" of a target protein is preferably a polypeptide or peptide that comprises a portion of the full-length ammo acid sequence of the reference protein. A fragment will retain at least one functional, structural, or antigenic characteristic of the parent protein, and more preferably retains at least one antigenic characteristic (for example, such that a target-binding protein described herein will specifically bind to the target protein and to a fragment of said target protein).

[0139] As used herein, the term "sequence identity" refers to the degree of similarity between two nucleic acid or amino acid sequences, expressed as a percentage. Sequence identity is determined by comparing two sequences over a defined region using a specified algorithm and alignment parameters. For nucleic acid sequences, sequence identity is calculated by aligning the sequences and determining the percentage of identical nucleotide bases (A, T, C, G) at corresponding positions. For protein sequences, sequence identity refers to the percentage of identical amino acid residues at aligned positions. Sequence identity may be determined using any standard alignment algorithm such as BLAST, ClustalW. or Needleman-Wunsch. with default parameters. Sequence alignment is calculated over the full length of the reference sequence, unless otherwise stated,

[0140] As used herein, a “vector’' or “recombinant vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Standard techniques may be used for recombinant DNA preparation, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzy matic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed Atorney Docket No. 45288-0513WO1 throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

[0141] Vascular Endothelial Growth Factor A (VEGF-A)

[0142] Vascular Endothelial Growth Factor A (VEGF-A) is a secreted, disulfide-linked homodimeric glycoprotein belonging to the VEGF / PDGF (platelet-derived growth factor) group of the cystine-knot superfamily of hormones and extracellular signaling molecules, which are all characterized by the presence of eight conserved cysteine residues forming the typical cystine-knot structure. VEGF-A plays a role as glycosylated mitogen that specifically acts on endothelial cells and has various effects, including mediating increased vascular permeability, inducing angiogenesis, vasculogenesis and endothelial cell growth, promoting cell migration, and inhibiting apoptosis. As used herein, the term “VEGF-A” encompasses the protein having the amino acid sequence of SEQ ID NO: 1 as well as isoforms, fragments and variants thereof. Alternatively spliced transcripts, encoding either freely secreted or cell- associated isoforms, have been characterized, e.g. splice isoforms of VEGF-A, e g., VEGF121, VEGF145, VEGF165, VEGFisg and VEGF206, together with the naturally occurring allelic and processed forms thereof. In some embodiments, fragments include but are not limited to the 110-amino acid human vascular endothelial cell growth factor generated by plasmin cleavage of VEGF165. In some embodiments, a VEGF-A protein can comprise the amino acid sequence of SEQ ID NO: 1, splice isoforms VEGF121, VEGF145, VEGFies, VEGFi89 and VEGF206, the 110-amino acid fragment thereof, variants of the amino acid sequence of SEQ ID NO: 1, variants of the splice isoforms VEGF121, VEGF145, VEGF165, VEGFisg and VEGF206, and variants of the 110-amino acid fragment thereof.

[0143] SEQ ID NO: 1 - Human VEGF-A

[0144] MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVALKLFV QLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQWRLGA RKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPPHSPSRRGSA SRAGPGRASETMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKF MDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNI TMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKK Atorney Docket No. 45288-0513WO1

[0145] SRYKSWSVPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKP

[0146] RR

[0147] Five human VEGF-A isoforms of 121, 145, 165, 189 or 206 amino acids in length (VEGF-A121-206), encoded by distinct mRNA splice variants, have been described, all of which are capable of stimulating mitogenesis in endothelial cells. These isoforms differ in biological activity, receptor specificity, and affinity for cell surface- and extracellular matrix-associated heparan-sulfate proteoglycans, which behave as low affinity receptors for VEGF-A. For example, VEGF-A121 does not bind to either heparin or heparan-sulfate; VEGF-A145 and VEGF-A165 are both capable of binding to heparin; and VEGF-A189 and VEGF-A206 show the strongest affinity for heparin and heparan-sulfates. VEGF-A121. VEGF-A145, and VEGF- Ai65 are secreted in a soluble form, although most of VEGF-A165 is confined to cell surface and extracellular matrix proteoglycans, whereas VEGF-Ai89 and VEGF-A206 remain associated with extracellular matrix. Both VEGF-A189 and VEGF-A206 can be released by treatment with heparin or heparinase, indicating that these isoforms are bound to extracellular matrix via proteoglycans. Cell-bound VEGF-Aiso can also be cleaved by proteases such as plasmin, resulting in release of an active soluble VEGF-A110.

[0148] Most tissues that express VEGF-A are observed to express several VEGF-A isoforms simultaneously, although VEGF-Ai2i and VEGF-Aies are the predominant forms, whereas VEGF-A2oe is rarely detected. VEGF-A145 differs in that it is primarily expressed in cells derived from reproductive organs. Human VEGF-A165, the most abundant and biologically active form, is glycosylated at Asn74 and is ty pically expressed as a 46 kDa homodimer of 23 kDa subunits.

[0149] Four cell-surface receptors that interact with VEGF-A have been identified. These include VEGFR-l / Flt-1 (fins-like tyrosine kinase- 1); VEGFR-2 / KDR / Flk-l (kinase insert domain containing receptor / fetal liver kinase-1); neuropilin-1, and neuropilin-2. VEGF121 and VEGF 165 bind VEGFR-1; VEGF121, VEGF145, and VEGFies bind VEGFR-2; VEGF165 binds neuropilin- 1; and VEGF165 and VEGF 145 bind neuropilin-2. The two best characterized VEGF receptors are VEGFR-l / Flt-1 and VEGFR-2 / KDR / Flk- 1. The specificity of each receptor for each VEGF family member varies but VEGF-A binds to both Flt-1 and KDR. The full length Flt-1 receptor includes an extracellular domain that has seven Ig domains, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. The extracellular domain is involved in the binding of VEGF and the intracellular domain is involved in signal transduction. VEGF-A receptor molecules, or fragments thereof, that specifically bind to VEGF-A can be Atorney Docket No. 45288-0513WO1 used as VEGF-A inhibitors that bind to and sequester the VEGF-A protein, thereby preventing it from signaling. Also the soluble form of the receptor exerts an inhibitory effect on the biological activity of the VEGF-A protein by binding to VEGF-A, thereby preventing it from binding to its natural receptors present on the surface of target cells.

[0150] Furthermore, VEGF-A-driven angiogenesis has a major role in the pathogenesis of diverse human diseases, including cancer, eye disorders, and rheumatoid arthritis. Recognition of the importance of VEGF-A for the development of several important classes of cancer recently culminated in the approval of AVASTIN™, a humanized monoclonal antibody to VEGF-A, for the treatment of metastatic colorectal cancer. Similarly, the importance of VEGF- A in the pathogenesis of neovascular ocular disorders is reflected in the recent approval of LUCENTIS™. a humanized monoclonal antibody fragment, for the treatment of neovascular (wet) age-related macular degeneration (AMD). Thus, in some embodiments, VEGF-A targeting therapy can be used to treat patients with undesirable angiogenesis and vascular leakage in cancer and other VEGF-A associated diseases (e.g., inflammation, cardiovascular disease, or autoimmune disease).

[0151] Proteins that include a VEGF-A protein binding domain

[0152] Provided herein are proteins that include a VEGF-A protein binding domain, such as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. As used herein, a “VEGF- A protein binding domain” refers to a binding domain that binds specifically to a VEGF-A protein. In some embodiments, the protein comprises a sequence that is at least 80% (e g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, the protein is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0153] SEQ ID NO: 2 - GDM VEGFA 54

[0154] AEKKEKIIKALELLAEAAKKLEEAAEDPSLKEALKELKEKLKEIKEKLKKGEISLEDA ANQIGALGAMIIDFADGMLAMGKIDEAEEVLKLVKEAAKALIEGGGEAGRAGRSISA KIASLEKRIAAAK

[0155] SEQ ID NO: 3 - GDM VEGFA 79 Atorney Docket No. 45288-0513WO1

[0156] SIADIIALLEGVRDAVLAGNLDEALALMKKAADAILAEEPASPEAKALIDAAIAALEA

[0157] GDFDEADAKLAEASKLIEKEGGSLAAQVVVSAMLLLGVALKSNDPALIKGVANDIG

[0158] QLIDILKDWAASQ

[0159] SEQ ID NO: 4 - GDM VEGFA 66

[0160] TPEKELIEEAILALALGDREGAAAKLRELGELDPENKAFFEAQASTLLKSTNEDQLDG MMAVLLSYILEKFPLAEAEAFIEALADRVLASDAPLERKAAFLSIAASLLELEGGDPA LIARLRARAAELAAQAA

[0161] SEQ ID NO: 5 - GDM VEGFA 71

[0162] GPKIHEFEGSTPGVKVVAIIGGGHAVVIAEMDIPADPAKIAKAKAALEAKAKEIEARL APVLDRVTVHVAVDTSSNPPKAILVVELGGADAERVERLALELAKDLLEFLEKLAKE LNP

[0163] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 2. In some embodiments, the protein is SEQ ID NO: 2.

[0164] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 3. In some embodiments, the protein is SEQ ID NO: 3.

[0165] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 4. In some embodiments, the protein is SEQ ID NO: 4.

[0166] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 5. In some embodiments, the protein is SEQ ID NO: 5.

[0167] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%. at least 99%. or 100%) identical to SEQ ID NO: 2. In some embodiments, the protein is SEQ ID NO: 2. Atorney Docket No. 45288-0513WO1

[0168] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 3. In some embodiments, the protein is SEQ ID NO: 3.

[0169] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%. at least 99%. or 100%) identical to SEQ ID NO: 4. In some embodiments, the protein is SEQ ID NO: 4.

[0170] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%. at least 99%. or 100%) identical to SEQ ID NO: 5. In some embodiments, the protein is SEQ ID NO: 5.

[0171] In some embodiments, the proteins can be single-chain polypeptides. In some embodiments, the protein can have a total number of amino acids of about 50 amino acids to about 140 amino acids (e.g.. about 50 amino acids to about 120 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 120 amino acids, or about 120 amino acids to about 140 amino acids).

[0172] In some embodiments, the protein binds to the extracellular domain of VEGF-A. In some embodiments, the protein binding to VEGF-A inhibits VEGF-A activity. As used herein, the term ‘'VEGF-A activity” refers to a variety of functions of the VEGF-A protein, including pro-angiogenic activity, vascular permeability activity7, and the stimulation of cell migration in macrophage lineage and endothelial cells. In some embodiments, the protein binding to VEGF- A neutralizes VEGF-A activity. In some embodiments, the protein binding to VEGF-A reduces VEGF-A activity.

[0173] In some embodiments, the protein binds to VEGF-A with a dissociation equilibrium constant (KD) of about 1 x 10’13M to about 1 x 10’8M. In some embodiments, the protein binds to VEGF-A with a dissociation equilibrium constant (KD) of less than 1 x 10’8M. less than 1 x 10-9M. less than 1 x 1010M, less than 1 x 10’11M, less than 1 x 1012M, or less than 1 x 10’13M. In some embodiments, the protein binds to VEGF-A with a KD value of about 1 x 10’8M to Atorney Docket No. 45288-0513WO1 about 1 x IO’10M, about 1 x 10’9M to about 1 x 10'11M, about 1 x 10'10M to about 1 x 10’12M, about 1 x 10’11M to about 1 x 10'13M.

[0174] A variety of different methods known in the art can be used to determine the KD values of any of the polypeptides described herein (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.).

[0175] Nucleic Acids

[0176] Also provided herein are nucleic acids that include a sequence that encodes any of the proteins described herein.

[0177] Also provided herein are vectors that include a sequence that encode any of the proteins described herein. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and viral derived vectors (e.g.. any adenoviral derived vectors (AV), cytomegaloviral derived (CMV) vectors, simian viral derived (SV40) vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors). In some embodiments, the expression vector is a viral vector.

[0178] Some embodiments of any of the vectors or nucleic acids described herein can further include a promoter operably linked to a sequence or sequences encoding the protein.

[0179] Also provided herein are cells including a nucleic acid encoding any of the proteins described herein, or a vector comprising any of the nucleic acids described herein. The cells may be non-human cells. The cells may be somatic cells. The cells may be autologous or heterologous cells.

[0180] Pharmaceutical Compositions and Therapeutic Applications

[0181] Also provided herein are pharmaceutical compositions that include at least one of any of the proteins described herein or any of the cells described herein. Also provided herein are pharmaceutical compositions that include at least one of any of the nucleic acids described herein or any of the vectors described herein. As used herein, the term “pharmaceutical composition’7refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers.

[0182] Also provided herein are methods of treating a VEGF-A associated disease in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of any of the proteins or pharmaceutical compositions described herein. Atorney Docket No. 45288-0513WO1

[0183] As used herein, the term “subject” refers to an organism, A pically a mammal (e.g., a human). In some embodiments, the subject is a human.

[0184] In some embodiments, the VEGF-A associated disease is a cancer, an inflammatory disease, a cardiovascular disease, or an autoimmune disease.

[0185] In some embodiments, the VEGF-A associated disease is cancer.

[0186] Non-limiting examples of such cancers are solid tumors and hematological cancers that include but are not limited to sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B- cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL). acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

[0187] In some embodiments, the cancer is colorectal cancer, lung cancer, glioblastoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, or renal cell cancer.

[0188] In some embodiments, the VEGF-A associated disease is a cardiovascular disease. Nonlimiting examples of cardiovascular diseases are coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, or deep vein thrombosis and pulmonary embolism.

[0189] In some embodiments, the cardiovascular disease is ischemic heart disease, heart failure, myocardial infarction, or coronary artery disease.

[0190] In some embodiments, the VEGF-A associated disease is an autoimmune disease. Nonlimiting examples of autoimmune diseases are psoriatic arthritis, rheumatoid arthritis (RA), Sjogren’s syndrome, systemic lupus ery thematosus (lupus, SLE), Crohn’s disease, celiac disease, ulcerative colitis, Graves’ disease, Hashimoto's thyroiditis, Addison’s disease, dermatomyositis, psoriasis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Bane syndrome, multiple sclerosis (MS), myasthenia gravis, autoimmune vasculitis, type 1 diabetes, pernicious anemia, or vasculitis.

[0191] In some embodiments, the autoimmune disease comprises systemic lupus erythematosus, rheumatoid arthritis, or multiple sclerosis.

[0192] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein Atorney Docket No. 45288-0513WO1 comprising SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity- to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0193] Some embodiments provide a method of treating cancer in a subject previously identified or diagnosed as having cancer, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 2, SEQ ID NO: 3. SEQ ID NO: 4, or SEQ ID NO: 5, or a protein having at least 80% (e.g., at least 85%, 90%, 95%. 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0194] In some embodiments, the subject has a clinical record indicating a diagnosis of cancer.

[0195] In some embodiments, the subject is suspected of having or at risk of developing cancer.

[0196] Some embodiments provide a method of treating cardiovascular disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. or SEQ ID NO: 5, or a protein having at least 80% (e.g., at least 85%. 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity- to SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4, or SEQ ID NO: 5.

[0197] Some embodiments provide a method of treating cardiovascular disease in a subject previously identified or diagnosed as having cardiovascular disease, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. or SEQ ID NO: 5, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity- to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0198] In some embodiments, the subject has a clinical record indicating a diagnosis of cardiovascular disease.

[0199] In some embodiments, the subject is suspected of having or at risk of developing cardiovascular disease.

[0200] Some embodiments provide a method of treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4. or SEQ ID NO: 5, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0201] Some embodiments provide a method of treating autoimmune disease in a subject previously identified or diagnosed as having an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID Atorney Docket No. 45288-0513WO1

[0202] NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%. 97%. 98%. 99% or 100%) sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0203] In some embodiments, the subject has a clinical record indicating a diagnosis of an autoimmune disease.

[0204] In some embodiments, the subject is suspected of having or at risk of developing an autoimmune disease.

[0205] In some embodiments, a protein as described herein is provided for use in therapy, and / or for use in any specific therapy described herein. Also provided is a use of a protein as described herein for manufacturing a medicament for treatment of any specific condition described herein.

[0206] Epstein-Barr Virus BCL-2 homolog (BHRF1) protein

[0207] Apoptosis and cell survival are regulated by the homeostatic balance of B cell lymphoma-2 (Bcl-2) family proteins, which fall into three classes. The ‘executioners’, Bak and Bax, initiate apoptosis by increasing mitochondrial outer membrane permeability and facilitating the release of mitochondrial cytochrome c to the cytosol, which activates downstream signaling. Six human pro-survival Bcl-2 proteins (Bcl-2, Bcl-XL, Bcl-B, Mcl-1, Bcl-w and Bfl-1) inhibit this process. Counterbalancing these are numerous pro-apoptotic BH3-only proteins (BOPs), including Bim. These factors share an approximately 26 residue Bcl-2 homology 3 (BH3) motif, an amphipathic a-helical element which binds a hydrophobic groove on the surface of the canonical Bcl-2 fold. Cellular stresses activate pro-apoptotic BOPs, which bind and inhibit pro-survival Bcl-2 members, and directly interact wi th Bak and Bax to favor mitochondrial permeabilization. Conversely, pro-survival Bcl-2 proteins dampen apoptotic triggers and enhance chemoresistance by sequestering BOPs or directly inhibiting Bak and Bax. Increased expression of pro-survival Bcl-2 proteins is a common feature of many cancers.

[0208] BHRF1 is a 17 kDa putative transmembrane protein, which is highly conserved among different EBV isolates and shows strong functional homology7with the human BCL2. This 191- amino-acid viral protein owns two motifs referred to as BCL2 homology domains 1 and 2 (BH1 and BH2). BHRF 1 anti-apoptotic activity may at least in part rely on its ability to interact with pro-apoptotic BH3-only proteins (e g., BCL2L11) and with the executioner BAKE wherein BHRF1, like BCL2, seems to reside mainly in the mitochondrial membrane. As used herein, Atorney Docket No. 45288-0513WO1 the term “BHRFT' encompasses the protein having the amino acid sequence of SEQ ID NO: 6 as well as, isoforms, fragments and variants thereof.

[0209] SEQ ID NO: 6 - Human BHRF1

[0210] MAYSTREILLALCIRDSRVHGNGTLHPVLELAARETPLRLSPEDTVVLRYHVLLEEIIE RNSETFTETWNRFITHTEHVDLDFNSVFLEIFHRGDPSLGRALAWMAWCMHACRTL CCNQSTPYYVVDLSVRGMLEASEGLDGWIHQQGGWSTLIEDNIPGSRRFSWTLFLAG LTLSLLVICSYLFISRGRH

[0211] BHRF1, an immediate early EBV antigen with sequence homology to Bcl-2, has a proven ability to act as a cell survival gene. In some studies, EBV-negative B-lymphoma (BL) cells expressing BHRF1 were rendered more resistant to programmed cell death (apoptosis) under experimental conditions of growth factor withdrawal and rodent hamster fibroblasts expressing BHRF1 displayed increased resistance to the apoptotic inducing effects of DNA- damaging drugs. These studies suggest that whilst BHRF1 is not consistently expressed in EBV-associated tumours, it is possible that expression of this protein at an early stage in the oncogenic process may influence the development of these malignancies. To date, the only in vivo lesion where BHRF1 is abundantly expressed is oral ‘hairy7’ leukoplakia (HL), a benign lesion of oral tongue mucosa which represents a focus of chronic EBV replication with absence of detectable latent gene expression.

[0212] BHRF1 belongs to an increasing family of viral proteins with sequence and functional homology7to Bcl-2 including the adenovirus E1B 19K protein and the recently described ORF 16 gene of Kaposi's sarcoma herpesvirus. Amongst this family of genes, the principal regions of homology are clustered within two motifs referred to as Bcl-2 homology domains 1 and 2 (BH1 and BH2). More specifically, the amino acid residues Gly-145 in BH1 and Trp- 188 in BH2 (Bcl-2 coordinates) have been demonstrated to be essential for the anti-apoptotic function of Bcl-2 and its ability7to heterodimerize with Bax. Furthermore, outside of BH1 and BH2, three further conserved homology7domains have been identified. NH1 and NH2 were identified by sequence comparison with the adenovirus E1B 19K protein and the Bcl-2 sequence, wherein domain swapping studies between the NH1 homology domain of adenovirus El B-19K protein and Bcl-2 have shown complementation of function. Analysis of Bik, another Bcl-2 homologue, has identified another domain, adjacent to NH1, referred to as BH3, which is present only in cell death-inducing proteins such as Bax and Bak and mediates the interaction of Bax with E1B-19K and Bcl-2 proteins. An interaction betw een the cell death-inducing Atorney Docket No. 45288-0513WO1 protein Bik and the cell death-suppressing proteins BHRF1 and E1B-19K has been demonstrated, and recent work indicates that BHRF 1 does not heterodimerize with Bax but can interact with p23 R- Ras, a member of the Ras superfamily that has previously been shown to interact with Bcl-2. Specific mutations in BHRF1 which abrogate the interaction with R-Ras confer a proliferative capacity on BHRF1. Thus, it was suggested that the R-Ras interaction normally acts to restrain this proliferative effect of BHRF1 and that mutations in BHRF 1 which prevent R-Ras binding may contribute to the pathogenesis of EBV-associated tumors.

[0213] Proteins that include a BHRF1 protein binding domain

[0214] Provided herein are proteins that include a BHRF1 protein binding domain, such as SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. As used herein, a BHRF I protein binding domain” refers to a binding domain that binds specifically to a BHRF1 protein. In some embodiments, the protein comprises a sequence that is at least 80% (e.g.. at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some embodiments, the protein is SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0215] SEQ ID NO: 7 - GDM BHRF1 70

[0216] MPSAFQIGLALVAAALDRALPEPYRGLALAIAAELSGLPEEELRRLVEAAEKAASAD LPFEQQVGLALARIAAAVAGVGLARRAPSLPPEELLAAIREAIEEGGRIAAKALTRSG ALEPVLAELP

[0217] SEQ ID NO: 8 - GDM BHRF1 35

[0218] KEEGRKLLEEAERALRLAEELLEQGRLEAAIPPLREAILLAVKAAELGLEEEALPLLD RAADLAERGAKKARERGDKKLALEFEVLAGVALIARGVALVALRNAK

[0219] SEQ ID NO: 9 - GDM BHRF1 72

[0220] KEKEREQKAVSLIAAAGIALAGLEFAPQPSAEELASVLELLEEAAALSTSEEDLAFLR RLAERARELLASLPDPPAELVARLEALLARLA

[0221] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at Atorney Docket No. 45288-0513WO1 least 98%, at least 99%, or 100%) identical to SEQ ID NO: 7. In some embodiments, the protein is SEQ ID NO: 7.

[0222] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 8. In some embodiments, the protein is SEQ ID NO: 8.

[0223] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 9. In some embodiments, the protein is SEQ ID NO: 9.

[0224] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 7. In some embodiments, the protein is SEQ ID NO: 7.

[0225] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g.. at least 85%. at least 90%. at least 92%. at least 94%. at least 95%. at least 96%. at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 8. In some embodiments, the protein is SEQ ID NO: 8.

[0226] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g.. at least 85%. at least 90%. at least 92%. at least 94%. at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 9. In some embodiments, the protein is SEQ ID NO: 9.

[0227] In some embodiments, the proteins can be single-chain polypeptides. In some embodiments, the protein can have a total number of amino acids of about 80 amino acids to about 120 amino acids (e.g., about 80 amino acids to about 1 10 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 90 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 100 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 110 amino acids, or about 110 amino acids to about 120 amino acids).

[0228] In some embodiments, the protein binding to BHRF1 inhibits BHRF1 activity. As used herein, the term “BHRF1 activity” refers to a variety of functions of the BHRF1 protein, including modifying mitochondrial dynamics, stimulating DNMIL / Drpl -mediated mitochondrial fission, pro-autophagic activity, and anti-apoptotic activity. In some Atorney Docket No. 45288-0513WO1 embodiments, the protein binding to BHRF1 neutralizes BHRF1 activity. In some embodiments, the protein binding to BHRF1 reduces BHRF1 activity.

[0229] In some embodiments, the protein binds to BHRF1 with a dissociation equilibrium constant (KD) of about 1 x 10'13M to about 1 x 10'8M. In some embodiments, the protein binds to BHRF1 with a dissociation equilibrium constant (KD) of less than 1 x 10'8M, less than 1 x 10’9M, less than 1 x 10'10M, less than 1 x 10’11M, less than 1 x 10'12M, or less than 1 x 10’13M. In some embodiments, the protein binds to BHRF 1 with a KD value of about 1 x 10'8M to about 1 x IO’10M, about 1 x 10'9M to about 1 x 10'11M, about 1 x 10'10M to about 1 x 10'12M, about 1 x 10’11M to about 1 x 10'13M.

[0230] A variety of different methods known in the art can be used to determine the KD values of any of the polypeptides described herein (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.).

[0231] Nucleic Acids

[0232] Also provided herein are nucleic acids that include a sequence that encodes any of the proteins described herein.

[0233] Also provided herein are vectors that include a sequence that encode any of the proteins described herein. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and viral derived vectors (e.g., any adenoviral derived vectors (AV), cytomegaloviral derived (CMV) vectors, simian viral derived (SV40) vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors). In some embodiments, the expression vector is a viral vector.

[0234] Some embodiments of any of the vectors or nucleic acids described herein can further include a promoter operably linked to a sequence or sequences encoding the protein.

[0235] Also provided herein are cells including a nucleic acid encoding any of the proteins described herein, or a vector comprising any of the nucleic acids described herein. The cells may be non-human cells. The cells may be somatic cells. The cells may be autologous or heterologous cells.

[0236] Pharmaceutical Compositions and Therapeutic Applications

[0237] Also provided herein are pharmaceutical compositions that include at least one of any of the proteins described herein or any of the cells described herein. Also provided herein are Atorney Docket No. 45288-0513WO1 pharmaceutical compositions that include at least one of any of the nucleic acids described herein or any of the vectors described herein.

[0238] Also provided herein are methods of treating a BHRF1 associated disease in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of any of the proteins or pharmaceutical compositions described herein.

[0239] In some embodiments, the BHRF 1 associated disease is a cancer.

[0240] Non-limiting examples of such cancers are solid tumors and hematological cancers that include but are not limited to sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B- cell lymphoma, B-cell non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, chronic lymphocytic leukemia (CLL). acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung carcinoma, squamous cell head and neck carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

[0241] In some embodiments, the cancer is non-Hodgkin B-cell lymphoma, T-cell lymphoma, or EBV-associated lymphoma.

[0242] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0243] Some embodiments provide a method of treating cancer in a subject previously identified or diagnosed as having cancer, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0244] In some embodiments, the subject has a clinical record indicating a diagnosis of cancer. In some embodiments, the subject is suspected of having or at risk of developing cancer. In some embodiments, a protein as described herein is provided for use in therapy, and / or for use in any specific therapy described herein. Also provided is a use of a protein as described herein for manufacturing a medicament for treatment of any specific condition described herein. Atorney Docket No. 45288-0513WO1

[0245] Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) Receptor Binding Domain (RBD)

[0246] Coronaviruses (CoV) are enveloped viruses with a positive-stranded RNA genome. SARS coronavirus 2 (SARS-CoV-2) is a highly transmissible and virulent coronavirus that infects host cells by binding to their common receptor, angiotensin converting enzyme 2 (ACE2), with their respective spike (S) protein. A discrete ~197-amino-acid domain of the S protein, referred to as the receptor-binding domain (RBD), directly associates with ACE2. The viral genome of SARS-CoV-2 encodes spike (S), envelope (E), membrane (M), and nucleocapsid (N) structural proteins, among which the S glycoprotein is responsible for binding the host receptor via the receptor-binding domain (RBD) in its SI subunit, as well as the subsequent membrane fusion and viral entry driven by its S2 subunit.

[0247] In some embodiments, the RBD is the major, if not the sole, neutralizing epitope on the SARS-CoV-2 spike (S) protein, and it elicits more neutralizing antibodies than the whole S protein. However, while RBD has been the focus of SARS-CoV-2 vaccine development, monomeric RBD is unlikely to make a potent vaccine because of its small size, its inability to crosslink the B-cell receptor or activate complement, or to stay bound in follicular dendritic cells in the lymph node. As used herein, the term “SARS_CoV-2 RBD” encompasses the protein having the amino acid sequence of SEQ ID NO: 10 as well as, isoforms, fragments and variants thereof.

[0248] SEQ ID NO: 10 -SARS-CoV-2 RBD

[0249] NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKV GGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGENCYFPLQSYGFQPTNG VGYQPYRVVVLSFELLHAPATVCGP

[0250] SARS-COV-2 infects epithelial cells of the respiratory tract, causing typical signs such as fever, dry cough, fatigue and dyspneal. A small fraction of patients however progresses towards a severe form of pneumonia requiring hospitalization or in some cases intensive care unit treatments. Cell entry of SARS-CoV-2 Coronavirus is mediated by the Spike glycoprotein that is present in multiple copies creating an extensive crown on the virus envelope. Spike glycoprotein is a class 1 fusion protein produced as a single polypeptide of about 1300 amino acids that trimerizes upon folding. It comprises two functional subunits called SI and S2: SI Atorney Docket No. 45288-0513WO1 represents the apex of the trimer and by its receptor binding domain (RBD). including residues from 331 to 524 of the S protein, promotes the attachment and fusion of virus particles with host membranes; S2 subunit, anchored to the viral membrane, contains a hydrophobic fusion loop and two heptad repeat regions (HR1 and HR2). Each S domain is around 20 nm surface projection that surrounds the periphery of the coronavirus and varies considerably between different coronaviruses. Within the S domain of SARS-CoV, there is a short domain (within the SI subunit) containing just 2 glycosylation sites that secrete short fragments of RBD (by glycosylation) that fold and bind to the ACE2 receptor.

[0251] The SARS-CoV-2 RBD has a twisted 5-stranded antiparallel beta-sheet with short connecting helices and loops. In the core, between the 04-7 strands, there is an additional extended insertion containing short 05-6 strands. This extension is where the receptor-binding motif (RBM) is, which contains the contacting residues that enable it to bind to ACE2. The interaction of Spike with the host receptor Angiotensin-Converting Enzy me 2 (ACE2) represents the first step of the infection, followed by proteolysis and conformational changes of Spike translating into the exposure of the fusion loop and its insertion into the target cell membrane with the release of viral genome into the cells. Consequently, the RBD of S 1 subunit is critical for determining host specificity and cell tropism.

[0252] Thus, in some embodiments, the identification of the RBD can help lead to the development of SARS-CoV-2 RBD binding compounds in the treatment and prevention of coronaviral infection, as the RBD is the site for many major neutralizing targeted therapies - preventing the virus binding to the receptor (ACE2 / DPP4).

[0253] Proteins that include a SARS-CoV-2 RBD protein binding domain

[0254] Provided herein are proteins that include a SARS-CoV-2 RBD protein binding domain, such as SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. As used herein, aL‘SARS-CoV-2 RBD protein binding domain” refers to a binding domain that binds specifically to a SARS-CoV-2 RBD protein. In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15. or SEQ ID NO: 16. In some embodiments, the protein is SEQ ID NO: 11. SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. Atorney Docket No. 45288-0513WO1

[0255] SEQ ID NO: 11 - GDM SC2RBD 104

[0256] MATATLTLDKTSAKPGDTITASATGSGTATIAGARVFVVLLAFDENGNQVDSASGSA

[0257] APGETATASLTVPAGCSKVKAFAGYGDPGANKGYITDWGTVEVT

[0258] SEQ ID NO: 12 - GDM SC2RBD 50

[0259] MSAVEKAIENAKKGLENAKKDGASEESIRGLKSAINLLKEYKEGVLPESLKADAEDL IKYFSAVKD

[0260] SEQ ID NO: 13 - GDM SC2RBD 143

[0261] EAIEEAGRRAEEIENPDVRGAASLALGAIYAQVKNGGTGGVTAAVAVAAVANGASP SLSDEELETVARFIVDALKLLGIELPSAETLREELEAVRKAMAHSMTPEELALFDRLA DALLAEVAA

[0262] SEQ ID NO: 14 - GDM SC2RBD 11

[0263] AAEADITLGSIIQSPSGTFAVVGGTAPAGTFPAEPTEALVKFHDGTVYHTGVTPMAM TDGTQNFSTVVPAEEAEASIGKTVTVTAGGGTVVGTLKRDPNLQVINL

[0264] SEQ ID NO: 15 - GDM SC2RBD 129

[0265] MATATLDAPEAAPIGTTVSATITGAPEGSTIFVTIVNLDTGLPVGSGSIRAASGTVSATI EGAKPGERYLAAAGYAADGSPVGTITAAKEFTVVE

[0266] SEQ ID NO: 16 - GDM SC2RBD 27

[0267] GNRLLAQFAGEATLEVDGETVYKGEGGFGVHDLNGRGVVTTGFNLTPEQAAKVSG TGWGTAKLVADGKEIASGPTGLVYDEESNILGANLLLSPEQAAAAGKAKTGKLEVE GTVGGKAVKMVAKGGLAESGDIPLGETA

[0268] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100%) identical to SEQ ID NO: 11. In some embodiments, the protein is SEQ ID NO: 11.

[0269] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100%) identical to SEQ ID NO: 12. In some embodiments, the protein is SEQ ID NO: 12. Atorney Docket No. 45288-0513WO1

[0270] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 13. In some embodiments, the protein is SEQ ID NO: 13.

[0271] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100%) identical to SEQ ID NO: 14. In some embodiments, the protein is SEQ ID NO: 14.

[0272] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100%) identical to SEQ ID NO: 15. In some embodiments, the protein is SEQ ID NO: 15.

[0273] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100%) identical to SEQ ID NO: 16. In some embodiments, the protein is SEQ ID NO: 16.

[0274] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 11. In some embodiments, the protein is SEQ ID NO: 11.

[0275] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 12. In some embodiments, the protein is SEQ ID NO: 12.

[0276] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 13. In some embodiments, the protein is SEQ ID NO: 13.

[0277] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 14. In some embodiments, the protein is SEQ ID NO: 14.

[0278] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least Atorney Docket No. 45288-0513WO1

[0279] 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 15. In some embodiments, the protein is SEQ ID NO: 15.

[0280] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 16. In some embodiments, the protein is SEQ ID NO: 16.

[0281] In some embodiments, the proteins can be single-chain polypeptides. In some embodiments, the protein can have a total number of amino acids of about 50 amino acids to about 140 amino acids (e.g., about 50 amino acids to about 120 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 120 amino acids, or about 120 amino acids to about 140 amino acids). In some embodiments, the protein can have a total number of amino acids of about 80 to about 120 amino acids.

[0282] In some embodiments, the protein binds to the extracellular domain of SARS-CoV-2 RBD. In some embodiments, the protein binding to SARS-CoV-2 RBD inhibits SARS-CoV-2 activity. As used herein, the term “SARS-CoV-2 activity” refers to a variety of functions of the SARS-CoV-2 protein, including facilitating the incorporation of the virus into the host cell, binding to the human angiotensin-converting enzyme 2 (ACE2) and thereby causing COVID- 19. In some embodiments, the protein binding to SARS-CoV-2 RBD neutralizes SARS-CoV- 2 activity. In some embodiments, the protein binding to SARS-CoV-2 RBD reduces SARS- CoV-2 activity.

[0283] In some embodiments, the protein binds to SARS-CoV-2 RBD with a dissociation equilibrium constant (KD) of about 1 x 10"13M to about 1 x 10'8M. In some embodiments, the protein binds to SARS-CoV-2 RBD with a dissociation equilibrium constant (KD) of less than 1 x 10’8M. less than 1 x 10‘9M, less than 1 x 10‘10M. less than 1 x 10'11M, less than 1 x 10’12M, or less than 1 x 10‘13M. In some embodiments, the protein binds to SARS-CoV-2 RBD with a KD value of about 1 x 10'8M to about 1 x IO'10M, about 1 x 10'9M to about 1 x 10'11M, about 1 x 10'10M to about 1 x IO'12M, about 1 x 10’11M to about 1 x 10'13M. Atorney Docket No. 45288-0513WO1

[0284] A variety of different methods known in the art can be used to determine the KD values of any of the polypeptides described herein (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.).

[0285] Nucleic Acids

[0286] Also provided herein are nucleic acids that include a sequence that encodes any of the proteins described herein.

[0287] Also provided herein are vectors that include a sequence that encode any of the proteins described herein. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and viral derived vectors (e.g., any adenoviral derived vectors (AV), cytomegaloviral derived (CMV) vectors, simian viral derived (SV40) vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors). In some embodiments, the expression vector is a viral vector.

[0288] Some embodiments of any of the vectors or nucleic acids described herein can further include a promoter operably linked to a sequence or sequences encoding the protein.

[0289] Also provided herein are cells including a nucleic acid encoding any of the proteins described herein, or a vector comprising any of the nucleic acids described herein. The cells may be non-human cells. The cells may be somatic cells. The cells may be autologous or heterologous cells.

[0290] Pharmaceutical Compositions and Therapeutic Applications

[0291] Also provided herein are pharmaceutical compositions that include at least one of any of the proteins described herein or any of the cells described herein. Also provided herein are pharmaceutical compositions that include at least one of any of the nucleic acids described herein or any of the vectors described herein.

[0292] Also provided herein are methods of treating a SARS-CoV-2 associated disease in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of any of the proteins or pharmaceutical compositions described herein.

[0293] In some embodiments, the SARS-CoV-2 associated disease is COVID19.

[0294] Some embodiments provide a method of treating COVID19 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15. or SEQ ID NO: 16, or a protein having at least 80% (e.g., at least 85%, 90%, 95%. 96%. Atorney Docket No. 45288-0513WO1

[0295] 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0296] Some embodiments provide a method of treating COVID 19 in a subject previously identified or diagnosed as having COVID 19, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0297] In some embodiments, the subject has a clinical record indicating a diagnosis of COVID19.

[0298] In some embodiments, the subject is suspected of having or at risk of developing COVID19.

[0299] In some embodiments, a protein as described herein is provided for use in therapy, and / or for use in any specific therapy described herein. Also provided is a use of a protein as described herein for manufacturing a medicament for treatment of any specific condition described herein.

[0300] Interleukin-7 receptor subunit alpha (IL7R-a) protein

[0301] The Interleukin-7 receptor subunit alpha (IL7R-a) also known as CD 127, together with IL2R-y (the common y chain) subunit forms a heterodimer receptor for Interleukin-7 (IL-7). The engagement of IL-7 with its receptor activates signal transduction via the JAK1 and JAK3 tyrosine kinases, which leads to dimerization of STAT3, STAT5A, and STAT5B transcription factors, activation of the PI3 kinase pathway and up-regulation of Bcl-2. IL7R-a is a Cytokine Receptor Homology class I (CRH I) receptor. As is well known in the art, the extracellular domain of these receptors consists of two fibronectin 3 domains, termed DI and D2. The precise crystallographic structure of CD127 has been published and discussed in e.g., McElroy et al., 2009; McElroy et al., 2012 and Walsh, 2012, wherein DI is generally considered to be involved in the binding with IL-7, while D2 is involved in the binding to the yc chain (and also with IL-7).

[0302] As used herein, the term "IL7R-a“ encompasses a protein having the amino acid sequence of SEQ ID NO: 17 as well as, isoforms, fragments and variants thereof. Atorney Docket No. 45288-0513WO1

[0303] SEQ ID NO: 17 - Human IL7R-U

[0304] MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTC AFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLT CKKIDLTTIVKPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEK DENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPE INNSSGEMDPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKP RKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSP NCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLL LSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0305] IL7Ra is common to the IL-7 receptor (IL7R). wherein the IL7R is constituted of a heterodimer of CD127 and the common gamma chain (yc) of interleukin receptors. In some embodiments, the common gamma chain yc is referred to as CD 132. In some embodiments, IL7R-a may exist in four isoforms. The canonical isoform is a single-pass transmembrane protein and has 459 amino acids consisting, from N- to C-terminal, of a 20 amino-acid signal peptide, a 219 ammo acid extracellular domain, a 25 amino-acid transmembrane domain and a 195 amino-acid intracellular domain. Other isoforms share the sequence of all of (or most of) the extracellular domain of the canonical isofrom and display varied C-terminal sequences. Isoforms 2 and 4 are secreted, while isoform 3 is also a transmembrane protein.

[0306] IL7R-a, which is expressed in lymphoid progenitors and therefore is important for IL- 7 signaling, is subsequently involved in several processes, including cell survival and proliferation during lymphoid development and in generation and maintenance of thymus- derived yoT cells. In humans, IL-7 signaling plays a critical role in the development of aPT cells, while in mice it is reported to be involved in the development of both T and B cells. Furthermore, it was shown that the IL7R-a signals are involved in the VDJ recombination process both in T and B cells, where the combinatorial joining of the V, D, and J segments encodes for the variable regions of the T cell receptor (TCR) in T cells and B cell receptor (BCR, i.e., Immunoglobulins) in B cells. This allows for the generation of the diversity and plasticity of the adaptive immune system which is critical for the development of T and B cells. Specifically, since the TCRy chain undergoes rearrangement prior to the TCR chain, TCRy (TRG) genes rearrangement are directly affected by IL-7 signaling, as was shown in IL7R- a- / - mice that harbor severe impairment in the y locus rearrangement. However, the direct effect of IL-7 signaling on the rearrangement of other loci (i.e., TCR , TCRa, TCR5) is still Atorney Docket No. 45288-0513WO1 unknown. Altogether, IL7Ra delivers trophic signals that protect lymphoid progenitors from a death process and maintain the viability of cells during gene rearrangement.

[0307] Binding of IL-7 to IL7R triggers the activation of several signaling pathways, including the Janus kinases (JAK) -1 and -3, signal transducer and activator of transcription 5 (STAT5) and phosphatidylinositol 3-kinase (PI3-k). STAT1 and STAT3 pathways are reported to be activated, although they do not seem to be the main pathways. The activation of the STAT5 pathway is required for the induction of the anti-apoptotic protein Bcl-2 and the prevention of the entry of the pro-apoptotic protein Bax in the mitochondrion and thus for survival of thymic developing T cell precursors. The activation of the PI3-k pathway results in the phosphorylation and cytoplasmic retention of the pro-apoptotic protein Bad.

[0308] Furthermore, in some embodiments, several malignant cells have been shown to display IL7R. For example, this is the case for Sezary cutaneous lymphoma (60% of them), or childhood acute lymphoblastic leukemia in which about 15% of the cases develop gain-of- function mutation in IL7Ra, rendering these tumors partially IL-7 dependent.

[0309] In some embodiments, the depletion of T lymphocytes has been an obvious immunosuppressive approach to counteract allograft rejection or fight autoimmunity. However, total T cell depletion might not be favorable for the induction of immunological tolerance. Therefore, targeting T cell subpopulations or selectively activated T cells, without modifying Treg cells, could constitute a pro-tolerogenic approach. IL7R-a may thus be regarded as a potential attractive therapeutic target, wherein it has been assumed accordingly that IL7R-a targeted therapies might show efficacy in transplantation, autoimmunity and malignancies by antagonizing access of IL-7 to IL7R and thereby limiting T and B cell function and growth.

[0310] Proteins that Include an IL7 Ra protein binding domain

[0311] Provided herein are proteins that include an IL7R-a protein binding domain, such as SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. As used herein, a “IL7R-a protein binding domain” refers to a binding domain that binds specifically to a IL7R-a protein. In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In some embodiments, the protein is SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

[0312] SEQ ID NO: 18 - GDM IL7RA 70 Atorney Docket No. 45288-0513WO1

[0313] MTKVEEAKELVDKIMEAAKAKDLEKVNKLRTEFFELVNSLSLEEAEEVRKYADKKG

[0314] EEWYKEQL

[0315] SEQ ID NO: 19 - GDM IL7RA 5

[0316] AVEPVLSKEEVGEIARIYAKEIGKDYGIELSDEEIDLAAELARELYGKSPEEAKEFLEE VYKKLSKELSKETLKIIIAAAVGALEAAELAGRLAEEYRAGVIDADELREELSKFLPD ELVDRVLARAEA

[0317] SEQ ID NO: 20 - GDM IL7RA 83

[0318] KTLLELADEFHEAVENKEYDKALAILDEIRKKYPEYKEGVDEARKRVEALKP

[0319] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 18. In some embodiments, the protein is SEQ ID NO: 18.

[0320] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 19. In some embodiments, the protein is SEQ ID NO: 19.

[0321] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 20. In some embodiments, the protein is SEQ ID NO: 20.

[0322] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 18. In some embodiments, the protein is SEQ ID NO: 18.

[0323] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g.. at least 85%. at least 90%. at least 92%. at least 94%. at least 95%. at least 96%. at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 19. In some embodiments, the protein is SEQ ID NO: 19.

[0324] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least Atorney Docket No. 45288-0513WO1

[0325] 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 20. In some embodiments, the protein is SEQ ID NO: 20.

[0326] In some embodiments, the proteins can be single-chain polypeptides. In some embodiments, the proteins can be multi-chain polypeptides. In some embodiments, the protein can have a total number of amino acids of about 50 amino acids to about 140 amino acids (e.g., about 50 amino acids to about 120 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 80 ammo acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 120 amino acids, or about 120 amino acids to about 140 amino acids). In some embodiments, the protein can have a total number of amino acids of about 50 amino acids to about 120 amino acids.

[0327] In some embodiments, the protein binding to IL7Ra inhibits IL7Ra activity. As used herein, the term “IL7Ra activity’’ refers to a variety of functions of the IL7Ra protein, including development of T cells, promoting immune effector functions in T lymphocytes, natural killer (NK) cells and monocytes macrophages, and modulating the quantity and quality of immune responses in vitro and in vivo. In some embodiments, the protein binding to IL7Ra neutralizes IL7Ra activity. In some embodiments, the protein binding to IL7Ra reduces IL7Ra activity.

[0328] In some embodiments, the protein binds to IL7Ra with a dissociation equilibrium constant (KD) of about 1 x 10'13M to about 1 x 10'8M. In some embodiments, the protein binds to IL7Ra with a dissociation equilibrium constant (KD) of less than 1 x 10'8M, less than 1 x 10-9M. less than 1 x 10-1° M, less than 1 x 10’11M, less than 1 x 1012M, or less than 1 x 10’13M. In some embodiments, the protein binds to IL7Ra with a KD value of about 1 x 10'8M to about 1 x 10’10M, about 1 x 10'9M to about 1 x 10'11M, about 1 x 10'10M to about 1 x 10'12M, about 1 x 10'11M to about 1 x 10'13M.

[0329] A variety of different methods known in the art can be used to determine the KD values of any of the polypeptides described herein (e.g.. an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.).

[0330] Nucleic Acids Atorney Docket No. 45288-0513WO1

[0331] Also provided herein are nucleic acids that include a sequence that encodes any of the proteins described herein.

[0332] Also provided herein are vectors that include a sequence that encode any of the proteins described herein. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and viral derived vectors (e.g., any adenoviral derived vectors (AV), cytomegaloviral derived (CMV) vectors, simian viral derived (SV40) vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors). In some embodiments, the expression vector is a viral vector.

[0333] Some embodiments of any of the vectors or nucleic acids described herein can further include a promoter operably linked to a sequence or sequences encoding the protein.

[0334] Also provided herein are cells including a nucleic acid encoding any of the proteins described herein, or a vector comprising any of the nucleic acids described herein. The cells may be non-human cells. The cells may be somatic cells. The cells may be autologous or heterologous cells.

[0335] Pharmaceutical Compositions and Therapeutic Applications

[0336] Also provided herein are pharmaceutical compositions that include at least one of any of the proteins described herein or any of the cells described herein. Also provided herein are pharmaceutical compositions that include at least one of any of the nucleic acids described herein or any of the vectors described herein.

[0337] Also provided herein are methods of treating an IL7R-A associated disease in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of any of the proteins or pharmaceutical compositions described herein.

[0338] In some embodiments, the IL7R-A associated disease is cancer.

[0339] Non-limiting examples of such cancers are solid tumors and hematological cancers such as melanoma, glioblastoma, esophagus tumor, nasopharyngeal carcinoma, uveal melanoma, lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, diffuse large B-cell lymphoma, primary' mediastinal large B-cell lymphoma, prostate cancer, castrationresistant prostate cancer, chronic myelocytic leukemia, Kaposi's sarcoma fibrosarcoma, liposarcoma. chondrosarcoma, osteogenic sarcoma, angiosarcoma, lymphangiosarcoma, synovioma, meningioma, leiomyosarcoma, rhabdomyosarcoma, sarcoma of soft tissue, sarcoma, sepsis, biliary tumor, basal cell carcinoma, thymus neoplasm, cancer of the thyroid Atorney Docket No. 45288-0513WO1 gland, cancer of the parathyroid gland, uterine cancer, cancer of the adrenal gland, liver infection, Merkel cell carcinoma, nerve tumor, follicle center lymphoma, colon cancer, Hodgkin's disease, non-Hodgkin's lymphoma, leukemia, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, ovary tumor, myelodysplastic syndrome, cutaneous or intraocular malignant melanoma, renal cell carcinoma, small-cell lung cancer, lung cancer, mesothelioma, breast cancer, squamous non-small cell lung cancer (SCLC), non-squamous NSCLC, colorectal cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, pancreatic cancer, Pancreatic ductal adenocarcinoma, squamous cell carcinoma of the head and neck, cancer of the head or neck, gastrointestinal tract, stomach cancer, bone cancer, skin cancer, rectal cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the urethra, cancer of the penis, cancer of the bladder, cancer of the kidney, cancer of the ureter, carcinoma of the renal pelvis, spinal axis tumor, brain stem glioma, pituitary adenoma, epidermoid cancer, asbestosis, carcinoma, adenocarcinoma, papillary carcinoma, cystadenocarcinoma, bronchogenic carcinoma, renal cell carcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embry onal carcinoma, Wilm’s tumor, pleomorphic adenoma, liver cell papilloma, renal tubular adenoma, cystadenoma, papilloma, adenoma, leiomyoma, rhabdomyoma, hemangioma, lymphangioma, osteoma, chondroma, lipoma and fibroma.

[0340] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 18. SEQ ID NO: 19. or SEQ ID NO: 20, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

[0341] Some embodiments provide a method of treating cancer in a subject previously identified or diagnosed as having cancer, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

[0342] In some embodiments, the subject has a clinical record indicating a diagnosis of cancer.

[0343] In some embodiments, the subject is suspected of having or at risk of developing cancer. Atorney Docket No. 45288-0513WO1

[0344] In some embodiments, a protein as described herein is provided for use in therapy, and / or for use in any specific therapy described herein. Also provided is a use of a protein as described herein for manufacturing a medicament for treatment of any specific condition described herein.

[0345] Programmed death-ligand 1 (PD-L1) protein

[0346] Programmed death-ligand 1 (PD-L1) (also referred to as CD274 or B7-H1) is a transmembrane protein that performs a major role in suppressing the adaptive immune system during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states. PD-L1 is also expressed on the neoplastic cells of many different cancers. By binding to PD-1 on T-cells, PD-L1 expression is a major mechanism by which tumor cells can evade immune attack. As used herein, the term "PD-L I " encompasses a protein having the amino acid sequence of SEQ ID NO: 21 as well as, isoforms, fragments and variants thereof.

[0347] SEQ ID NO: 21 - Human PD-LI

[0348] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVY WEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVY RCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTS SDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPE LPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDT HLEET

[0349] PD-LI is a member of the B7 family of immunoregulatory ligands that play a pivotal role in regulation of the cellular and humoral immune responses. PD-LI is expressed in both lymphoid and non-lymphoid tissue, where it is involved in suppression of immune activity through activation of the co-inhibitory receptor programmed cell death protein- 1 (PD-1), which is expressed primarily on T cells, but also some natural killer (NK) cells and monocytes. Immunosuppressive pathways (also called immune checkpoint pathways) such as the PD- 1 / PD-L1 axis are essential for maintaining healthy immune regulation. However, these pathways are frequently exploited by cancer cells and immune cells in the tumor microenvironment to evade immune recognition. Indeed, PD-LI expression is observed in various forms of cancer, both constitutively and as a feedback response to inflammatory signals, resulting in inhibition of tumor specific T cell responses. Atorney Docket No. 45288-0513WO1

[0350] Blockade of the PD-1 / PD-L1 pathway restores functionality to chronically exhausted CD8+ T cells, suggesting that tumor-associated PD-L1 primarily inhibits proliferation and effector functions of activated tumor-specific T cells. Upregulation of PD-L1 by tumor cells is often associated with adaptive resistance to endogenous tumor-specific immune responses, in particular the secretion of interferon-y (IFN-y) as well as inflammatory cytokines. Although high-level expression of PD-L1 in tumor biopsies has generally been used as a criterion to guide anti-PD-Ll treatment, it has become increasingly clear that PD-L1 expression on infiltrating immune cells, including myeloid cells and T cells, can in many cases be sufficient to predict therapeutic efficacy. Some data suggest that PD-L1 expression on antigen-presenting cells may play a vital role in determining the efficacy of anti-PD-Ll therapy, whereas PD-L1 on tumor cells and tumor infiltrating lymphocytes is irrelevant, highlighting the continued uncertainty on the underlying mechanisms of PD-L1 -targeted therapies. Unfortunately, patient response rates to anti-PD-Ll therapies remain disappointingly low and most tumor regressions are only partial, necessitating the development of new mechanistic strategies to enhance the efficacy of immune checkpoint protein targeted therapies.

[0351] PD-L1 over-expression may conceptually be due to two mechanisms, intrinsic and adaptive. Intrinsic expression of PD-L1 on cancer cells is related to cellular / genetic aberrations in these neoplastic cells. Activation of cellular signaling including the AKT and STAT pathways results in increased PD-L1 expression. In primary mediastinal B-cell lymphomas, gene fusion of the MHC class II trans activator (CUT A) with PD-L1 or PD-L2 occurs, resulting in overexpression of these proteins. Amplification of chromosome 9p23-24, where PD-L1 and PD-L2 are located, leads to increased expression of both proteins in classical Hodgkin lymphoma. Adaptive mechanisms are related to induction of PD-L1 expression in the tumor microenvironment. PD-L1 can be induced on neoplastic cells in response to interferon -y. In microsatellite instability colon cancer, PD-L1 is mainly expressed on myeloid cells in the tumors, which then suppress cytotoxic T-cell function.

[0352] In a tumor microenvironment, programmed death ligand 1 (PD-L1) can perform a vital role in tumor progression and survival by escaping tumor neutralizing immune surveillance. Enhancing T cell activation by blocking the PD-1 and PD-L1 inhibitory pathway has shown beneficial anti-tumor responses and long-term remissions in a subset of patients with a broad spectrum of cancers. Therefore, use of an inhibitor that blocks the interaction of PD-L1 with the PD-1 can help prevent PD-1 stimulation (e.g., on T cells), thereby increasing T cell function signals and immune cell responses. Atorney Docket No. 45288-0513WO1

[0353] Proteins that include a PD-L1 protein binding domain

[0354] Provided herein are proteins that include a PD-L 1 protein binding domain, such as SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. As used herein, a “PD-L1 protein binding domain” refers to a binding domain that binds specifically to a PD-L1 protein. In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In some embodiments, the protein is SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0355] SEQ ID NO: 22 - GDM PDL1 135

[0356] SAEEKILANLEAMKAKALAAKTEEEKLFYAKALLAVAISYAIRGDYELARRAAELAV EVIKSLSKEEQKKVMDFLINIIKNITDPEDREKAIELAIAIAERLDEEVREEALKKIEEL KKE

[0357] SEQ ID NO: 23 - GDM PDL1 142

[0358] SKAEAAANRMKRFLDGLKISIPELRDLIEKYGEKIVEAIKAGDKEKALKYAEELAKKI KEVLTDDPVFAENLAKFVIVYVESLLEEL

[0359] SEQ ID NO: 24 - GDM PDL1 138

[0360] LKEEALELADEVIKLAEELGWKDHVKAVEALKEAVEKSTDERFLASAKAFLEVLKE VLLEEKKA

[0361] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100%) identical to SEQ ID NO: 22. In some embodiments, the protein is SEQ ID NO: 22.

[0362] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100%) identical to SEQ ID NO: 23. In some embodiments, the protein is SEQ ID NO: 23.

[0363] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at Atorney Docket No. 45288-0513WO1 least 98%, at least 99%. or 100%) identical to SEQ ID NO: 24. In some embodiments, the protein is SEQ ID NO: 24.

[0364] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 22. In some embodiments, the protein is SEQ ID NO: 22.

[0365] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 23. In some embodiments, the protein is SEQ ID NO: 23.

[0366] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 24. In some embodiments, the protein is SEQ ID NO: 24.

[0367] In some embodiments, the proteins can be single-chain polypeptides. In some embodiments, the protein can have a total number of amino acids of about 50 amino acids to about 140 amino acids (e.g., about 50 amino acids to about 120 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 120 amino acids, or about 120 amino acids to about 140 amino acids). In some embodiments, the protein can have a total number of amino acids of about 50 amino acids to about 120 amino acids.

[0368] In some embodiments, the protein binding to PD-L1 inhibits PD-L1 activity. As used herein, the term “PD-L1 activity” refers to a variety of functions of the PD-L1 protein, including inhibiting immune responses and promoting self-tolerance through modulating the activity of T-cells, activating apoptosis of antigen-specific T cells and inhibiting apoptosis of regulatory T cells, reducing the proliferation of PD-1 positive cells, inhibiting their cytokine secretion and inducing apoptosis. In some embodiments, the protein binding to PD-L1 neutralizes PD-L1 activity. In some embodiments, the protein binding to PD-L1 reduces PD- L1 activity. Atorney Docket No. 45288-0513WO1

[0369] In some embodiments, the protein binds to PD-L1 with a dissociation equilibrium constant (KD) of about 1 x 10’13M to about 1 x 10’8M. In some embodiments, the protein binds to PD-L1 with a dissociation equilibrium constant (KD) of less than 1 x 10'8M, less than 1 x 10'9M, less than 1 x 10'10M, less than 1 x 10'11M, less than 1 x 10'12M, or less than 1 x 10'13M. In some embodiments, the protein binds to PD-L1 with a KD value of about 1 x 10'8M to about 1 x IO’10M, about 1 x 10’9M to about 1 x 10'11M, about 1 x 10'10M to about 1 x 10’12M, about 1 x 10’11M to about 1 x 10'13M.

[0370] A variety of different methods known in the art can be used to determine the KD values of any of the polypeptides described herein (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.).

[0371] Nucleic Acids

[0372] Also provided herein are nucleic acids that include a sequence that encodes any of the proteins described herein.

[0373] Also provided herein are vectors that include a sequence that encode any of the proteins described herein. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and viral derived vectors (e.g.. any adenoviral derived vectors (AV), cytomegaloviral derived (CMV) vectors, simian viral derived (SV40) vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors). In some embodiments, the expression vector is a viral vector.

[0374] Some embodiments of any of the vectors or nucleic acids described herein can further include a promoter operably linked to a sequence or sequences encoding the protein.

[0375] Also provided herein are cells including a nucleic acid encoding any of the proteins described herein, or a vector comprising any of the nucleic acids described herein. The cells may be non-human cells. The cells may be somatic cells. The cells may be autologous or heterologous cells.

[0376] Pharmaceutical Compositions and Therapeutic Applications

[0377] Also provided herein are pharmaceutical compositions that include at least one of any of the proteins described herein or any of the cells described herein. Also provided herein are pharmaceutical compositions that include at least one of any of the nucleic acids described herein or any of the vectors described herein. Atorney Docket No. 45288-0513WO1

[0378] Also provided herein are methods of treating aPD-Ll associated disease in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of any of the proteins or pharmaceutical compositions described herein.

[0379] In some embodiments, the PD-L1 associated disease is a cancer, an inflammatory disease, an infectious disease, or an autoimmune disease.

[0380] In some embodiments, the PD-L1 associated disease is cancer.

[0381] Non-limiting examples of such cancers are solid tumors and hematological cancers such as melanoma, glioblastoma, esophagus tumor, nasopharyngeal carcinoma, uveal melanoma, lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, diffuse large B-cell lymphoma, primary' mediastinal large B-cell lymphoma, prostate cancer, castrationresistant prostate cancer, chronic myelocytic leukemia, Kaposi's sarcoma fibrosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, lymphangiosarcoma, synovioma, meningioma, leiomyosarcoma, rhabdomyosarcoma, sarcoma of soft tissue, sarcoma, sepsis, biliary' tumor, basal cell carcinoma, thymus neoplasm, cancer of the thyroid gland, cancer of the parathyroid gland, uterine cancer, cancer of the adrenal gland, liver infection, Merkel cell carcinoma, nerve tumor, follicle center lymphoma, colon cancer. Hodgkin's disease, non-Hodgkin's lymphoma, leukemia, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, ovary tumor, myelodysplastic syndrome, cutaneous or intraocular malignant melanoma, renal cell carcinoma, small-cell lung cancer, lung cancer, mesothelioma, breast cancer, squamous non-small cell lung cancer (SCLC), non-squamous NSCLC, colorectal cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, pancreatic cancer, Pancreatic ductal adenocarcinoma, squamous cell carcinoma of the head and neck, cancer of the head or neck, gastrointestinal tract, stomach cancer, bone cancer, skin cancer, rectal cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the urethra, cancer of the penis, cancer of the bladder, cancer of the kidney, cancer of the ureter, carcinoma of the renal pelvis, spinal axis tumor, brain stem glioma, pituitary adenoma, epidermoid cancer, asbestosis, carcinoma, adenocarcinoma, papillary' carcinoma, cystadenocarcinoma, bronchogenic carcinoma, renal cell carcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm’s tumor, pleomorphic adenoma, liver cell papilloma, renal tubular adenoma, cystadenoma, papilloma, Atorney Docket No. 45288-0513WO1 adenoma, leiomyoma, rhabdomyoma, hemangioma, lymphangioma, osteoma, chondroma, lipoma and fibroma.

[0382] In some embodiments, the PD-L1 associated disease is an infectious disease. Nonlimiting examples of infectious diseases include viral diseases, bacterial diseases, and fungal diseases, such as HIV, Hepatitis A, Hepatitis B, Hepatitis C, hepatitis D, herpes viruses, papillomaviruses, influenza, herpes, giardia, malaria, leishmania. herpes virus (e.g., N N, HSV-I. HAV-6. HSV-II. and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus, pathogenic infection by the bacteria chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, E. coli, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme's disease bacteria, pathogenic infection by the fungi Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger. etc.), Genus Mucorales (mucor. absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis , Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum, and pathogenic infection by the parasites Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii. Plasmodium vivax. Babesia microti. Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani. Toxoplasma gondi, and Nippostrongylus brasiliensis.

[0383] In some embodiments, the infection is a viral infection. In some embodiments, the infection is a bacterial infection. In some embodiments, the infection is a fungal infection.

[0384] In some embodiments, the PD-L1 associated disease is an autoimmune disease. Nonlimiting examples of autoimmune diseases are systemic lupus erythematosis, rheumatoid arthritis, spondyloarthropathies, Sjogren’s syndrome, Guillain-Barre syndrome, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis (including (Grave’s disease. Hashimoto’s thyroiditis, juvenile lymphocytic thyroiditis, and atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, psoriasis, psoriatic arthritis, ankylosing spondylitis, asthma, allergic rhinitis, atopic dermatitis, and graft rejection.

[0385] In some embodiments, the autoimmune disease is systemic lupus erythematosus, psoriasis, rheumatoid arthritis, or multiple sclerosis. Atorney Docket No. 45288-0513WO1

[0386] In some embodiments, the PD-L1 associated disease is an inflammatory' disease. Nonlimiting examples of inflammatory diseases are acute inflammation, chronic inflammation, osteoarthritis, juvenile chronic arthritis, systemic vasculitis, systemic sclerosis, idiopathic inflammatory myopathies, sarcoidosis, idiopathic demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, non-viral hepatitis, primary' biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, gluten-sensitive enteropathy, erythema multiforme, contact dermatitis, eosinophilic pneumonia, idiopathic pulmonary fibrosis, and graft-versus-host-disease.

[0387] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 22. SEQ ID NO: 23. or SEQ ID NO: 24, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0388] Some embodiments provide a method of treating cancer in a subject previously identified or diagnosed as having cancer, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 22, SEQ ID NO: 23. or SEQ ID NO: 24, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity' to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0389] In some embodiments, the subject has a clinical record indicating a diagnosis of cancer. In some embodiments, the subject is suspected of having or at risk of developing cancer. Some embodiments provide a method of treating an infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 22. SEQ ID NO: 23. or SEQ ID NO: 24, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0390] Some embodiments provide a method of treating an infectious disease in a subject previously identified or diagnosed as having an infectious disease, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 22, SEQ ID NO: 23. or SEQ ID NO: 24.

[0391] In some embodiments, the subject has a clinical record indicating a diagnosis of an infectious disease. Atorney Docket No. 45288-0513WO1

[0392] In some embodiments, the subject is suspected of having or at risk of developing an infectious disease.

[0393] Some embodiments provide a method of treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 22, SEQ ID NO: 23. or SEQ ID NO: 24.

[0394] Some embodiments provide a method of treating an autoimmune disease in a subject previously identified or diagnosed as having an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0395] In some embodiments, the subject has a clinical record indicating a diagnosis of an autoimmune disease.

[0396] In some embodiments, the subject is suspected of having or at risk of developing an autoimmune disease.

[0397] Some embodiments provide a method of treating an inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

[0398] Some embodiments provide a method of treating an inflammatory disease in a subject previously identified or diagnosed as having an inflammatory disease, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 22, SEQ ID NO: 23. or SEQ ID NO: 24.

[0399] In some embodiments, the subject has a clinical record indicating a diagnosis of an inflammatory disease.

[0400] In some embodiments, the subject is suspected of having or at risk of developing an inflammatory disease.

[0401] In some embodiments, a protein as described herein is provided for use in therapy, and / or for use in any specific therapy described herein. Also provided is a use of a protein as Atorney Docket No. 45288-0513WO1 described herein for manufacturing a medicament for treatment of any specific condition described herein.

[0402] Tropomyosin receptor kinase A (Trk-A) protein

[0403] Trk-A is a transmembrane protein, encoded by the NTRK1 gene located on chromosome Iq21-q22. The Trk-A receptor consists of an extracellular domain, a transmembrane region and an intracellular region containing the tyrosine kinase domain. The extracellular domain contains a cysteine-rich cluster (Cl) followed by three leucine-rich 24- residue repeats (LRR1-3), another cysteine-rich cluster (C2) and two immunoglobulin-like domains (Igl and Ig2). The LRR1-3 motifs are specific to Trk proteins and are not found in other receptor tyrosine kinases. The intracellular region contains five key tyrosine residues: three within the activation loop of the kinase domain, which are necessary for full kinase activity’, and two on either side of the tyrosine kinase domain, which serve as phosphorylationdependent docking sites for cytoplasmic adaptors and enzy mes. As used herein, the term “Trk- A” encompasses a protein having the amino acid sequence of SEQ ID NO: 25 as well as, isoforms, fragments and variants thereof.

[0404] SEQ ID NO: 25 - Human Trk-A

[0405] MLRGGRRGQLGWHSWAAGPGSLLAWLILASAGAAPCPDACCPHGSSGLRCTRDGA LDSLHHLPGAENLTELYIENQQHLQHLELRDLRGLGELRNLTIVKSGLRFVAPDAFHF TPRLSRLNLSFNALESLSWKTVQGLSLQELVLSGNPLHCSCALRWLQRWEEEGLGG VPEQKLQCHGQGPLAHMPNASCGVPTLKVQVPNASVDVGDDVLLRCQVEGRGLEQ AGWILTELEQSATVMKSGGLPSLGLTLANVTSDLNRKNVTCWAENDVGRAEVSVQ VNVSFPASVQLHTAVEMHHWCIPFSVDGQPAPSLRWLFNGSVLNETSFIFTEFLEPAA NETVRHGCLRLNQPTHVNNGNYTLLAANPFGQASASIMAAFMDNPFEFNPEDPIPVS FSPVDTNSTSGDPVEKKDETPFGVSVAVGLAVFACLFLSTLLLVLNKCGRRNKFGIN RPAVLAPEDGLAMSLHFMTLGGSSLSPTEGKGSGLQGHIIENPQYFSDACVHHIKRR DIVLKWELGEGAFGKVFLAECHNLLPEQDKMLVAVKALKEASESARQDFQREAELL TMLQHQHIVRFFGVCTEGRPLLMVFEYMRHGDLNRFLRSHGPDAKLLAGGEDVAPG PLGLGQLLAVASQVAAGMVYLAGLHFVHRDLATRNCLVGQGLVVKIGDFGMSRDI YSTDYYRVGGRTMLPIRWMPPESILYRKFTTESDVWSFGVVLWEIFTYGKQPWYQL SNTEAIDCITQGRELERPRACPPEVYAIMRGCWQREPQQRHSIKDVHARLQALAQAP PVYLDVLG Atorney Docket No. 45288-0513WO1

[0406] The tropomyosin receptor kinase (Trk) family is classified as receptor ty rosine kinases and comprises (i) Trk-A which is a high-affinity receptor of nerve growth factor (NGF), (ii) Trk-B which is a high-affinity receptor of brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-4 / 5, and (iii) Trk-C which is a high-affinity receptor of NT-3. All Trk receptors are highly expressed in nerve tissues and are involved in differentiation and maintenance of functions of nerve cells. Meanwhile it has been known that activation of Trk- A in peripheral nerves by NGF initiates hyperalgesia and based on clinical and non-clinical test results using anti-NGF antibodies and non-clinical test results using low-molecular weight Trk inhibitors, involvement of Trk-A has been reported in (i) nociceptive pain of osteoarthritis, chronic low back pain, rheumatoid arthritis, bone fracture, interstitial cystitis and chronic pancreatitis, and (ii) neuropathic pain as well as cancer pain combining both types of pain described above. As such, NGF signaling via Trk-A is recognized to play an important role in pain sensation. For example, genetic studies in humans with Trk-A loss of function mutations have provided evidence of the significant role of NGF signaling in pain sensation, wherein NGF expression is increased in various pain conditions and administration of NGF increases pain sensitivity. Furthermore, inhibition of NGF signaling via Trk-A using a variety of antibody and small molecule-based approaches have been shown to be effective in preclinical animal models for pain.

[0407] Moreover, Trk receptors are expressed on cancer cells such as neuroblastoma, prostate cancer and pancreatic cancer, inflammatory cells such as mast cells and eosinophils, immunocompetent cells such as T cells and B cells and keratinocytes. Trk receptors are also reported to be potentially involved in proliferation, migration and metastasis of cancer cells, inflammatory' diseases such as ulcerative colitis and Crohn's disease, allergic diseases such as asthma, rhinitis and atopic dermatitis and other diseases such as psoriasis.

[0408] Trks play a key role in malignant transformation, chemotaxis, metastasis, and survival signaling in human tumors. In some embodiments, oncogenic activation of Trk-A occurs through genomic rearrangement and the creation of a gene fusion where extracellular domain of Trk-A is replaced by fusion with another gene with the kinase domain intact results in constitutive activation of Trk-A pathway. A number of NTRK1 gene fusions have been reported in a variety' of cancers that include, but are not limited to, such as NSCLC, spitz melanoma, colorectal cancer, cholangiocarcinoma, soft tissue sarcoma, glioblastoma and papillary thyroid carcinoma with more new fusions being reported based on the NGS sequencing of patient DNA. Atorney Docket No. 45288-0513WO1

[0409] In addition to gene fusions, molecular alterations such as an in-frame deletion of NTRK1 (ATrkA) in acute myeloid leukemia (AML) and a splice variant of NTRK1 (TRKAIII) in neuroblastoma have been functionally characterized as oncogenic. Autocrine and paracrine signaling by Trk receptors have been implicated as pro-tumorigenic in several different tumor types. For example, an autocrine loop involving Trk-A and NGF is associated with pro- tumorigenic activity in both breast and prostate carcinomas, and expression of Trk-A and TrkC wild-type receptors is associated with a positive prognosis in patients with neuroblastoma. Hence, TrkA inhibitors have potential for cancers driven by activated Trk-A signaling due to molecular alterations or autocrine / paracrine signaling due to increased expression of Trk-A and / or NGF.

[0410] NGF and Trk-A are expressed in immune cells and a localized increase in NGF at the sites of inflammation is observed during the inflammatory process. Inflammatory cytokines such as IL-lbeta, TNF-alpha and IL-6 are able to modify the basal production of NGF in the organism and induce the synthesis of NGF in a variety of cell ty pes and tissues. TrkA-NGF pathway is also involved in a number of disorders such as osteoarthritis, multiple sclerosis, inflammatory diseases (e.g.. asthma), interstitial cystitis, inflammatory bowel diseases (e.g.. ulcerative colitis, Crohn's disease), and neurodegenerative diseases (e.g., Alzheimer's disease, Huntington's disease, Progressive Supranuclear Palsy).

[0411] Trk kinases are also involved in skin diseases like atopic dermatitis, eczema, psoriasis, pruritis, restenosis, and atherosclerosis. In some embodiments, TrkA inhibition can also be implicated for the treatment of fibrotic disorders based on the ability of Connective Tissue Grow th Factor (CTGF) to activate Trk-A signaling. In some embodiments, inhibition of TrkA can also be useful in treatment of endometriosis, diabetic peripheral neuropathy, chronic prostatitis / chronic pelvic pain syndrome, and Chagas' disease. Hence, pharmacological inhibition of Trk-A pathway offers promising approaches for the treatment of a variety of diseases dependent on hyperactivation of Trk-A pathway.

[0412] Proteins that include a Trk-A protein binding domain

[0413] Provided herein are proteins that include a Trk-A protein binding domain, such as SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. As used herein, a ‘Trk-A protein binding domain” refers to a binding domain that binds specifically to a Trk-A protein. In some embodiments, the protein comprises a sequence that is at least 80% (e g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least Atorney Docket No. 45288-0513WO1

[0414] 99%, or 100%) identical to SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In some embodiments, the protein is SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

[0415] SEQ ID NO: 26 - GDM TrkA 9

[0416] APAPVLVDAGANVCKVTSGGKTSYRVLAVAGFQLPPGAGAPTVTSVTVTPHNGAA AVTIENVRAGTFSENGVTYAIVLGWAEIDAATAAALTGAPATVTVTADGKTYSKDV TIVASTATFTPA

[0417] SEQ ID NO: 27 - GDM TrkA 12

[0418] LELVSTNAPQPISGSLADGTAISGESSASVWTATESGDYPVKVTATNTGSGTVYGGGI VLAQNAGSDKLQGIGIGLTAIPPGKSVSNSGTLTVTKGGLIACAGSALCAEGGSGTLT NTITVGGKEVFSQTFTC

[0419] SEQ ID NO: 28 - GDM TrkA 130

[0420] SIVDELKEYFEEYKHHLSKQTKEAVEKGLADLEKILADPEKATTSEAYVFAVGAGAI AYAALKAGDKEKAEKVLELLEKVADSIPRESIRDTIRNAVRWIRRELEEYA

[0421] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100%) identical to SEQ ID NO: 26. In some embodiments, the protein is SEQ ID NO: 26.

[0422] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100%) identical to SEQ ID NO: 27. In some embodiments, the protein is SEQ ID NO: 27.

[0423] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100%) identical to SEQ ID NO: 28. In some embodiments, the protein is SEQ ID NO: 28.

[0424] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 26. In some embodiments, the protein is SEQ ID NO: 26. Atorney Docket No. 45288-0513WO1

[0425] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 27. In some embodiments, the protein is SEQ ID NO: 27.

[0426] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%. at least 99%. or 100%) identical to SEQ ID NO: 28. In some embodiments, the protein is SEQ ID NO: 28.

[0427] In some embodiments, the proteins can be single-chain polypeptides. In some embodiments, the protein can have a total number of amino acids of about 50 amino acids to about 140 amino acids (e.g.. about 50 amino acids to about 120 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 120 amino acids, or about 120 amino acids to about 140 amino acids). In some embodiments, the protein can have a total number of amino acids of about 50 amino acids to about 120 amino acids.

[0428] In some embodiments, the protein binding to Trk-A inhibits Trk-A activity. As used herein, the term “Trk-A activity” refers to a variety of functions of the Trk-A protein, including neuronal differentiation, neural proliferation, nociceptor response, and avoidance of programmed cell death. In some embodiments, the protein binding to Trk-A neutralizes Trk-A activity. In some embodiments, the protein binding to Trk-A reduces Trk-A activity.

[0429] In some embodiments, the protein binds to Trk-A with a dissociation equilibrium constant (KD) of about 1 x ICT13M to about 1 x 10'8M. In some embodiments, the protein binds to Trk-A with a dissociation equilibrium constant (KD) of less than 1 x 10'8M, less than 1 x 10’9M, less than 1 x 10’10M, less than 1 x 10'11M, less than 1 x 10'12M. or less than 1 x 10"13M. In some embodiments, the protein binds to Trk-A with a KD value of about 1 x 10‘8M to about 1 x 1 O'10M, about 1 x 1 O'9M to about 1 x 10'11M, about 1 x 1 O'10M to about 1 x 1 O’12M, about 1 x 10'11M to about 1 x 10'13M.

[0430] A variety of different methods known in the art can be used to determine the KD values of any of the polypeptides described herein (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.). Atorney Docket No. 45288-0513WO1

[0431] Nucleic Acids

[0432] Also provided herein are nucleic acids that include a sequence that encodes any of the proteins described herein.

[0433] Also provided herein are vectors that include a sequence that encode any of the proteins described herein. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and viral derived vectors (e.g., any adenoviral derived vectors (AV), cytomegaloviral derived (CMV) vectors, simian viral derived (SV40) vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors). In some embodiments, the expression vector is a viral vector.

[0434] Some embodiments of any of the vectors or nucleic acids described herein can further include a promoter operably linked to a sequence or sequences encoding the protein.

[0435] Also provided herein are cells including a nucleic acid encoding any of the proteins described herein, or a vector comprising any of the nucleic acids described herein. The cells may be non-human cells. The cells may be somatic cells. The cells may be autologous or heterologous cells.

[0436] Pharmaceutical Compositions and Therapeutic Applications

[0437] Also provided herein are pharmaceutical compositions that include at least one of any of the proteins described herein or any of the cells described herein. Also provided herein are pharmaceutical compositions that include at least one of any of the nucleic acids described herein or any of the vectors described herein.

[0438] Also provided herein are methods of treating a Trk-A associated disease in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of any of the proteins or pharmaceutical compositions described herein.

[0439] In some embodiments, the Trk-A associated disease is a cancer or an inflammatory disease.

[0440] In some embodiments, the Trk-A associated disease is cancer.

[0441] In some embodiments, the cancer is a solid tumor or a hematological cancer.

[0442] In some embodiments, the cancer is selected from the group of: adenocarcinoma, adrenal gland cortical carcinoma, adrenal gland neuroblastoma, anus squamous cell carcinoma, appendix adenocarcinoma, bladder urothelial carcinoma, bile duct adenocarcinoma, bladder carcinoma, bladder urothelial carcinoma, bone chordoma, bone marrow leukemia lymphocytic Atorney Docket No. 45288-0513WO1 chronic, bone marrow leukemia non-lymphocytic acute myelocytic, bone marrow lymph proliferative disease, bone marrow multiple myeloma, bone sarcoma, brain astrocytoma, brain glioblastoma, brain medulloblastoma, brain meningioma, brain oligodendroglioma, breast adenoid cystic carcinoma, breast carcinoma, breast ductal carcinoma in situ, breast invasive ductal carcinoma, breast invasive lobular carcinoma, breast metaplastic carcinoma, cervix neuroendocrine carcinoma, cervix squamous cell carcinoma, colon adenocarcinoma, colon carcinoid tumor, duodenum adenocarcinoma, endometrioid tumor, esophagus adenocarcinoma, esophagus and stomach carcinoma, eye intraocular melanoma, eye intraocular squamous cell carcinoma, eye lacrimal duct carcinoma, fallopian tube serous carcinoma, gallbladder adenocarcinoma, gallbladder glomus tumor, gastroesophageal junction adenocarcinoma, head and neck adenoid cystic carcinoma, head and neck carcinoma, head and neck neuroblastoma, head and neck squamous cell carcinoma, kidney chromophore carcinoma, kidney medullary carcinoma, kidney renal cell carcinoma, kidney renal papillary carcinoma, kidney sarcomatoid carcinoma, kidney urothelial carcinoma, kidney carcinoma, leukemia lymphocytic, leukemia lymphocytic chronic, liver cholangiocarcinoma. liver hepatocellular carcinoma, liver carcinoma, lung adenocarcinoma, lung adenosquamous carcinoma, lung atypical carcinoid, lung carcinosarcoma, lung large cell neuroendocrine carcinoma, lung nonsmall cell lung carcinoma, lung sarcoma, lung sarcomatoid carcinoma, lung small cell carcinoma, lung small cell undifferentiated carcinoma, lung squamous cell carcinoma, upper aerodigestive tract squamous cell carcinoma, upper aerodigestive tract carcinoma, lymph node lymphoma diffuse large B cell, lymph node lymphoma follicular lymphoma, lymph node lymphoma mediastinal B-cell, lymph node lymphoma plasmablastic lung adenocarcinoma, lymphoma follicular lymphoma, lymphoma, non-Hodgkins, nasopharynx and paranasal sinuses undifferentiated carcinoma, ovary carcinoma, ovary carcinosarcoma, ovary clear cell carcinoma, ovary epithelial carcinoma, ovary granulosa cell tumor, ovary serous carcinoma, pancreas carcinoma, pancreas ductal adenocarcinoma, pancreas neuroendocrine carcinoma, peritoneum mesothelioma, peritoneum serous carcinoma, placenta choriocarcinoma, pleura mesothelioma, prostate acinar adenocarcinoma, prostate carcinoma, rectum adenocarcinoma, rectum squamous cell carcinoma, skin adnexal carcinoma, skin basal cell carcinoma, skin melanoma, skin Merkel cell carcinoma, skin squamous cell carcinoma, small intestine adenocarcinoma, small intestine gastrointestinal stromal tumors (GISTs), large intestine / colon carcinoma, large intestine adenocarcinoma, soft tissue angiosarcoma, soft tissue Ewing sarcoma, soft tissue hemangioendothelioma, soft tissue inflammatory myofibroblastic tumor, soft tissue leiomyosarcoma, soft tissue liposarcoma, soft tissue neuroblastoma, soft tissue Atorney Docket No. 45288-0513WO1 paraganglioma, soft tissue perivascular epithelioid cell tumor, soft tissue sarcoma, soft tissue synovial sarcoma, stomach adenocarcinoma, stomach adenocarcinoma diffuse-type, stomach adenocarcinoma intestinal type, stomach adenocarcinoma intestinal type, stomach leiomyosarcoma, thymus carcinoma, thymus thymoma lymphocytic, thyroid papillary carcinoma, unknown primary adenocarcinoma, unknown primary7carcinoma, unknown primary7malignant neoplasm, lymphoid neoplasm, unknown primary melanoma, unknown primary sarcomatoid carcinoma, unknown primary7squamous cell carcinoma, unknown undifferentiated neuroendocrine carcinoma, unknown primary undifferentiated small cell carcinoma, uterus carcinosarcoma, uterus endometrial adenocarcinoma, uterus endometrial adenocarcinoma endometrioid, uterus endometrial adenocarcinoma papillary serous, and uterus leiomyosarcoma.

[0443] In some embodiments, the Trk-A associated disease is an inflammatory disease. Nonlimiting examples of inflammatory diseases are osteoarthritis, juvenile chronic arthritis, and inherited arthritis.

[0444] In some embodiments, the inflammatory disease is osteoarthritis.

[0445] In some embodiments, the inflammatory disease is juvenile chronic arthritis.

[0446] In some embodiments, the inflammatory disease is inherited arthritis.

[0447] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 26. SEQ ID NO: 27. or SEQ ID NO: 28.

[0448] Some embodiments provide a method of treating cancer in a subject previously identified or diagnosed as having cancer, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

[0449] In some embodiments, the subject has a clinical record indicating a diagnosis of cancer. In some embodiments, the subject is suspected of having or at risk of developing cancer. Some embodiments provide a method of treating an inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 26, SEQ ID NO: 27. or SEQ ID NO: 28. Atorney Docket No. 45288-0513WO1

[0450] Some embodiments provide a method of treating an inflammatory disease in a subject previously identified or diagnosed as having an inflammatory disease, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 26, SEQ ID NO: 27. or SEQ ID NO: 28.

[0451] In some embodiments, the subject has a clinical record indicating a diagnosis of an inflammatory disease.

[0452] In some embodiments, the subject is suspected of having or at risk of developing an inflammatory disease.

[0453] In some embodiments, a protein as described herein is provided for use in therapy, and / or for use in any specific therapy described herein. Also provided is a use of a protein as described herein for manufacturing a medicament for treatment of any specific condition described herein.

[0454] Interleukin- 17 (IL-17A) protein

[0455] The IL- 17 protein family contains six isoforms of 20-30 kDa molecular weight and is a group of secreted and glycosylated proteins. All other members of the IL-17 family show 20- 55% sequence homology to IL-17A, wherein IL-17A exists as a homodimer of two 136 amino acid chains that are secreted by activated T-cells. Structurally, IL-17A has a conserved C- terminus with four cysteine residues, which form intramolecular disulfide bridges. IL-17A is also known to act on stromal cells to induce production of proinflammatory and hematopoietic bioactive molecules. As used herein, the term “IL-17A” encompasses a protein having the amino acid sequence of SEQ ID NO: 29 as well as, isoforms, fragments and variants thereof.

[0456] SEQ ID NO: 29 - Human IL-17A

[0457] MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNT NPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVP IQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA

[0458] Although a signature cytokine of Thl7 cells. IL-17A is now known to be expressed also by other adaptive and immune cell types, including CD8+ T cells, y5 T cells, natural killer T (NKT) cells, and innate lymphoid cells. IL-17A was also shown to be expressed by mouse Atorney Docket No. 45288-0513WO1 neutrophils and, more recently, a population of human neutrophils was identified that expresses the transcription factor RORyt and both produces and responds to IL-17A. Consistent with a certain degree of inherent plasticity, naive T cells, memory T cells, and CD4+ Foxp3+ regulatory T cells (Tregs) have all been shown to have the ability to differentiate into an IL- 17-producing phenotype. The resulting IL-17-producing T cell can express varying concentrations of different effectors such as IL- 17 and IL- 10, potentially exhibiting either a pathogenic or regulatory phenotype.

[0459] IL-17 signals dominantly in non-hematopoietic cells to induce innate-like acute immune defenses. One hallmark function of IL-17 is induction of chemokines, including CXCL1, CXCL2 and CXCL8 (IL-8), that attract myeloid cells such as neutrophils, to infected or injured tissue. Additionally. IL- 17 induces IL-6 and G-CSF, cytokines that promote myeloid-driven innate inflammation. Together with induction of antimicrobial peptides such as b-defensins, S 100 A8 and lipocalin 2, these responses protect the host during acute microbial invasion. Accordingly, IL- 17 responses defend against extracellular fungal and bacterial pathogenic species including Candida, Cryptococcus, Klebsiella and Staphylococcus, among others. Indeed, genetic defects in the Thl7 or IL-17 signaling pathway in humans or in mice lead to severe mucocutaneous Candida infections in humans, which points to the particular importance of IL-17 in immunity to fungi.

[0460] IL- 17A proteins signal through a receptor complex consisting of IL- 17RA and IL- 17RC. which are present on many different cell types. IL-17A stimulates tissue-mediated innate immune responses that include promoting neutrophil recruitment, antimicrobial peptide production, and enhanced epithelial barrier function. These responses are especially important in mediating protective immunity against fungi and bacteria. Dysregulated expression of IL- 17 may contribute to inflammatory and autoimmune diseases such as psoriasis, psoriatic arthritis, rheumatoid arthritis, and multiple sclerosis. As such, they are highly interesting new therapeutic targets for inflammatory diseases.

[0461] Proteins that include an IL-17 A protein binding domain

[0462] Provided herein are proteins that include an IL-17A protein binding domain, such as SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. As used herein, an “IL-17A protein binding domain” refers to a binding domain that binds specifically to an IL-17A protein. In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at Atorney Docket No. 45288-0513WO1 least 99%, or 100%) identical to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. In some embodiments, the protein is SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

[0463] SEQ ID NO: 30 - GDM IL17A-57

[0464] SLLNEIRKILGEIDTIDAERFAGGDADSTPYIEKLEALVAAAPDEDLLDIARYLLELLTT PMSHDTEKAIARALIAALEKLVKKLGVKSEEIEELLERIRAAIERGEGLSGEQLDELGK ILNELELIHLASKS

[0465] SEQ ID NO: 31 - GDM IL17A-44

[0466] GKTVVVDPKVDEGAARAEAEKMAKDAAPDATLMGVIKVGIGSSGDSETITVTAPDG TSISVDIPVPAFHFSALWAAPGQPDRTLTVSKTVKVPGSLTLTQDGKTKTVDVDINVK ITVTGTVWDL

[0467] SEQ ID NO: 32 - GDM IL17A-52

[0468] SDEDWEFLKISGAKAALSNLAGIANMGFQAQLDALGDLLSAASPEVKAEAFRLIDDA QAAGVDVTPAVSLAIALAAKDLAAKGIPVNKDDLKALLDAALASVDKDLADPSKTD EQKAKLKEIKAKIEALAATI

[0469] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 30. In some embodiments, the protein is SEQ ID NO: 30.

[0470] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 31. In some embodiments, the protein is SEQ ID NO: 31.

[0471] In some embodiments, the protein comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. or 100%) identical to SEQ ID NO: 32. In some embodiments, the protein is SEQ ID NO: 32.

[0472] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 30. In some embodiments, the protein is SEQ ID NO: 30. Atorney Docket No. 45288-0513WO1

[0473] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 31. In some embodiments, the protein is SEQ ID NO: 31.

[0474] In some embodiments, the protein consists essentially of a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%. at least 99%. or 100%) identical to SEQ ID NO: 32. In some embodiments, the protein is SEQ ID NO: 32.

[0475] In some embodiments, the proteins can be single-chain polypeptides. In some embodiments, the protein can have a total number of amino acids of about 50 amino acids to about 140 amino acids (e.g.. about 50 amino acids to about 120 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 120 amino acids, or about 120 amino acids to about 140 amino acids).

[0476] In some embodiments, the protein binding to IL-17A inhibits IL-17A activity. As used herein, the term “IL-17A activity’” refers to a variety of functions of the IL-17A protein, including host defense activity, cell trafficking, immune modulation, tissue repair, induction of innate immune defense, and promoting chemokine production. In some embodiments, the protein binding to IL-17A neutralizes IL-17A activity'. In some embodiments, the protein binding to IL-17A reduces IL-17A activity’.

[0477] In some embodiments, the protein binds to IL-17A with a dissociation equilibrium constant (KD) of about 1 x ICT13M to about 1 x 10'8M. In some embodiments, the protein binds to IL-17A with a dissociation equilibrium constant (KD) of less than 1 x 10'8M, less than 1 x 10’9M, less than 1 x 10'10M, less than 1 x 10’11M, less than 1 x 10'12M, or less than 1 x 10’13M. In some embodiments, the protein binds to IL-17A with a KD value of about 1 x 10'8M to about 1 x 10’10M, about 1 x 10'9M to about 1 x 10'11M, about 1 x 10'10M to about 1 x 10'12M, about 1 x 10’11M to about 1 x 10'13M.

[0478] A variety of different methods known in the art can be used to determine the KD values of any of the polypeptides described herein (e.g., an electrophoretic mobility’ shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.). Atorney Docket No. 45288-0513WO1

[0479] Nucleic Acids

[0480] Also provided herein are nucleic acids that include a sequence that encodes any of the proteins described herein.

[0481] Also provided herein are vectors that include a sequence that encode any of the proteins described herein. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and viral derived vectors (e.g., any adenoviral derived vectors (AV), cytomegaloviral derived (CMV) vectors, simian viral derived (SV40) vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors). In some embodiments, the expression vector is a viral vector.

[0482] Some embodiments of any of the vectors or nucleic acids described herein can further include a promoter operably linked to a sequence or sequences encoding the protein.

[0483] Also provided herein are cells including a nucleic acid encoding any of the proteins described herein, or a vector comprising any of the nucleic acids described herein. The cells may be non-human cells. The cells may be somatic cells. The cells may be autologous or heterologous cells.

[0484] Pharmaceutical Compositions and Therapeutic Applications

[0485] Also provided herein are pharmaceutical compositions that include at least one of any of the proteins described herein or any of the cells described herein. Also provided herein are pharmaceutical compositions that include at least one of any of the nucleic acids described herein or any of the vectors described herein.

[0486] Also provided herein are methods of treating an IL- 17 associated disease in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of any of the proteins or pharmaceutical compositions described herein.

[0487] In some embodiments, the IL- 17 associated disease is a cancer, an inflammatory disease, an infection, or an autoimmune disease.

[0488] In some embodiments, the IL- 17 associated disease is cancer.

[0489] Non-limiting examples of such cancers are solid tumors and hematological cancers such as breast cancer, colon cancer, and colorectal cancer.

[0490] In some embodiments, the IL- 17 associated disease is an infection. Non-limiting examples of infections are viral infections such as hepatitis (e.g.. any of hepatitis A. B, C, D, Atorney Docket No. 45288-0513WO1 or E), herpes, and HIV, bacterial infections (such as Whipple’s disease), fungal infections, protozoal infections, and parasitic infections.

[0491] In some embodiments, the infection is a viral infection. In some embodiments, the viral infection is hepatitis, herpes, or HIV. In some embodiments, the infection is a bacterial infection. In some embodiments, the infection is a fungal infection. In some embodiments, the infection is a protozoal infection. In some embodiments, the infection is a parasitic infection.

[0492] In some embodiments, the IL-17 associated disease is an autoimmune disease. Nonlimiting examples of autoimmune diseases are systemic lupus erythematosis, rheumatoid arthritis, spondyloarthropathies, Sjogren’s syndrome, Guillain-Barre syndrome, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis (including (Grave’s disease. Hashimoto’s thyroiditis, juvenile lymphocytic thyroiditis, and atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, psoriasis, psoriatic arthritis, anky losing spondylitis, asthma, allergic rhinitis, atopic dermatitis, and graft rejection.

[0493] In some embodiments, the autoimmune disease is systemic lupus erythematosus, psoriasis, rheumatoid arthritis, or multiple sclerosis.

[0494] In some embodiments, the IL-17 associated disease is an inflammatory' disease. Nonlimiting examples of inflammatory diseases are acute inflammation, chronic inflammation, osteoarthritis, juvenile chronic arthritis, systemic vasculitis, systemic sclerosis, idiopathic inflammatory' myopathies, sarcoidosis, idiopathic demyelinating polyneuropathy, chronic inflammatory' demyelinating polyneuropathy, non-viral hepatitis, primary' biliary' cirrhosis, granulomatous hepatitis, sclerosing cholangitis, gluten-sensitive enteropathy, erythema multiforme, contact dermatitis, eosinophilic pneumonia, idiopathic pulmonary fibrosis, and graft-versus-host-disease.

[0495] Some embodiments provide a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 30. SEQ ID NO: 31. or SEQ ID NO: 32, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

[0496] Some embodiments provide a method of treating cancer in a subject previously identified or diagnosed as having cancer, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, Atorney Docket No. 45288-0513WO1

[0497] 98%, 99% or 100%) sequence identity' to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

[0498] In some embodiments, the subject has a clinical record indicating a diagnosis of cancer. In some embodiments, the subject is suspected of having or at risk of developing cancer. Some embodiments provide a method of treating an infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 30. SEQ ID NO: 31. or SEQ ID NO: 32, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

[0499] Some embodiments provide a method of treating an infection in a subject previously identified or diagnosed as having an infection, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

[0500] In some embodiments, the subject has a clinical record indicating a diagnosis of an infection.

[0501] In some embodiments, the subject is suspected of having or at risk of developing an infection.

[0502] Some embodiments provide a method of treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 30, SEQ ID NO: 31. or SEQ ID NO: 32.

[0503] Some embodiments provide a method of treating an autoimmune disease in a subject previously identified or diagnosed as having an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, or a protein having at least 80% (e.g., at least 85%. 90%. 95%. 96%. 97%. 98%, 99% or 100%) sequence identity to SEQ ID NO: 30. SEQ ID NO: 31, or SEQ ID NO: 32.

[0504] In some embodiments, the subject has a clinical record indicating a diagnosis of an autoimmune disease.

[0505] In some embodiments, the subject is suspected of having or at risk of developing an autoimmune disease. Atorney Docket No. 45288-0513WO1

[0506] Some embodiments provide a method of treating an inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

[0507] Some embodiments provide a method of treating an inflammatory disease in a subject previously identified or diagnosed as having an inflammatory disease, the method comprising administering to the subject a therapeutically effective amount of a protein comprising SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, or a protein having at least 80% (e.g., at least 85%, 90%, 95%, 96%. 97%. 98%, 99% or 100%) sequence identity to SEQ ID NO: 30, SEQ ID NO: 31. or SEQ ID NO: 32.

[0508] In some embodiments, the subject has a clinical record indicating a diagnosis of an inflammatory disease.

[0509] In some embodiments, the subject is suspected of having or at risk of developing an inflammatory’ disease.

[0510] In some embodiments, a protein as described herein is provided for use in therapy, and / or for use in any7specific therapy described herein. Also provided is a use of a protein as described herein for manufacturing a medicament for treatment of any specific condition described herein.

[0511] EXAMPLES

[0512] The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

[0513] A. Vascular Endothelial Growth Factor A (VEGF-A)

[0514] Methods

[0515] Target protein expression and purification

[0516] Purified protein stocks for VEGF-A(27-191) were purchased from BioTechne, with the catalog number AVI293. VEGF-A has a biotinylated C-terminal Avi tag and VEGF-A is a disulfide-linked homodimer. For x-ray crystallography, VEGF165 (Uniprot Pl 5692-4) was purchased from Qkine, with catalog number Qk048.

[0517] Yeast surface display and flow cytometry Atorney Docket No. 45288-0513WO1

[0518] Primary binding screen

[0519] Binder design sequences were codon-optimized by DNAworks and most were synthesized by Twist as gene fragments flanked by Bsal restriction sites as well as homology regions to a modified pETcon vector. Saccharomyces cerevisiae strain EBY100 cells (50 pL) were transformed using the lithium acetate method with 50 ng of linearized plasmid and a minimum of 10 ng of gene fragment insert in a 96-well plate. Cells were grown at 30 °C shaking at 1,000 rpm in complete synthetic medium -Trp -Ura + 2% glucose for 48-72 h. For protein expression, 2 x 107yeast cells were centrifuged at 1,800 x g for 5 mins at 20 °C and resuspended in 1 mb complete synthetic medium + 0.1% glucose + 2% galactose (SGCAA). Cells were incubated at 30 °C overnight (16-18 hours) and 8 x 106yeast cells were washed twice with 200 pL of lx PBS + 0.1% BSA (PBSF). centrifuged at 1,800 x g for 3 min at 20 °C and the supernatant was removed.

[0520] To screen for binding, yeast cells were then incubated with biotinylated target proteins (diluted in PBSF) for 1 hour, washed twice with PBSF and incubated with 25 ug / mL fluorescein isothiocyanate (FITC)-conjugated anti-Myc antibody (FITC-Ab) (Abeam) and 30 ug / mL streptavidin-phycoerythrin (SAPE, Thermo Fisher Scientific) for 30 minutes. For VEGF-A, the signal was increased by binding with avidity; target proteins were pre-incubated with 25 pg / mL FITC-Ab and 30 pg / mL SAPE for 30 minutes before incubating with cells. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. Cells were analyzed on the CytoFlex LX (Beckman Coulter) or ZE5 Cell Analyzer (Bio-Rad) flow cytometers by measuring fluorescence of FITC and phycoerythrin (PE) to detect binder expression and target binding respectively.

[0521] Interface mutation, competitive inhibition, and specificity experiments

[0522] Mutants were screened following the same method as the primary binding screen. For competition assays, yeast cells were incubated with biotinylated target proteins with or without a competitor protein for 1 h (the competitor protein was added to the biotinylated target protein master mix just before pipetting to the cells). The cells were then washed tw ice with PBSF and incubated with 25 pg / mL FITC-Ab (Abeam) and 30 pg / mL SAPE (Thermo Fisher Scientific) for 30 minutes. For VEGF-A, the signal was increased by binding with avidity similar to the primary binding screen; target proteins were pre-incubated with 25 pg / mL FITC-Ab and 30 pg / mL SAPE for 30 minutes before adding competitor protein and incubating with cells. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. Atorney Docket No. 45288-0513WO1

[0523] To test for specificity , specific designs as well as pooled cells were tested against target protein following the same method as the primary binding screen.

[0524] Designed binder expression and purification

[0525] Designed binders with the highest binding signal by yeast display were selected for E. coli expression and follow up experiments. Designs purchased as gene fragments were cloned into a modified pTriEx-4 vector containing an N-terminal 8-His tag using NEBndge Golden Gate cloning (NEB) at Bsal sites, transformed into DH5-a competent cells, miniprepped (Qiagen), and verified by Sanger sequencing (Azenta). A small number of designs were purchased from Twist Bioscience directly as cloned plasmids in pTriEx-4 or pET-29b. For expression, plasmids were transformed into BL21 (DE3) cells and the entire transformation mix inoculated into autoinduction medium consisting of Terrific Broth (TB) medium (Melford), 0.05% glucose, 0.2% alpha-lactose, and 50 p / mL carbenicillin. Cultures were incubated at 37 °C with shaking (220 or 1000 rpm) for 24 hours, harvested at 2,568 x g for 10 minutes, and pellets stored at -80 °C until purification. Cell pellets were lysed by chemical lysis using BugBuster Master Mix (Novagen) supplemented with complete EDTA-free protease inhibitor (Roche) with shaking for 20 minutes at room temperature. Lysates were clarified by centrifugation for 1 hour at 2,568 x g. then purified by immobilized metal affinity7chromatography (IMAC) using Ni-NTA in either 0. 1 mL spin columns (Cy tiva) or in HisPur™ Ni-NTA 96-well Spin Plates (Thermo Scientific), followed by size exclusion chromatography (SEC) on an AKTA Pure 25 M (Cytiva) equipped with an ALIAS autosampler (Spark Holland) using a Superdex 75 increase 10 / 300 GL column equilibrated in 20 mM sodium phosphate pH 7.5. Protein samples were analyzed by SDS-PAGE, and where required, concentrated using a 3 kDa MWCO Vivaspin concentrator (Cytiva). Protein concentrations were measured in triplicate by absorbance at 280 nm with a NanoDrop One (Thermo Scientific) using theoretical extinction coefficients, or using custom Python code. Purified proteins were aliquoted and stored at -80 °C until further use.

[0526] Where larger quantities of designed binders were required, for example for circular dichroism and x-ray crystallography experiments, expression was scaled up to 100-1000 mL BL21 (DE3) cultures and the above protocol followed with minor modifications. Cells were lysed using sonication for a total of 6 minutes (5 sec on / 5 sec off) on ice, and lysates were clarified by centrifugation at 40,000 x g for 45 minutes before being applied to 5 mL Ni-NTA column (Cytiva). followed by SEC. Atorney Docket No. 45288-0513WO1

[0527] Homogeneous Time Resolved Fluorescence (HTRF)

[0528] Measurement of binding affinity / binding dissociation constants (KD)

[0529] Binding affinities (KDS) were measured in equilibrium saturation-binding experiments with fixed binder design concentration and target titration. The total assay volume was 16 pL and all proteins and reagents w ere diluted in PPI europium detection buffer (Revvity). Target protein was premixed with HTRF acceptor reagent Streptavidin-d2 (Revvity), serially diluted, and transferred to a white ProxiPlate 384-shallow well microplate ("assay plate’. Revvity). Subsequently, 1 nM of each binder was added to the assay plate in duplicate (binders with KD < 0.5 nM were later re-assayed with 0.1 nM binder to ensure robust data fitting). The assay plate was centrifuged at 500 x g for 30 seconds, sealed and incubated at room temperature for between 30 minutes and 1 hour. HTRF donor mAb Anti-6HIS-Eu Gold (Revvity) was then added to a final concentration of 2 nM (lx), using a Mantis microfluidic liquid dispenser (Formulatrix) running software version 5.1.1 on Windows 10. The assay plate was centrifuged, sealed and incubated for a further 1 hour at room temperature.

[0530] HTRF signal was measured using a PHERAstar FSX (BMG) plate reader equipped with an HTRF 337 / 665 / 620 optic module running software version 5.70 R6 on Windows 10. The measurement conditions were as follows; 60 ps integration delay, 400 ps integration time, 60 flashes. The optimal focal (Z) height was determined using channel B for each experiment. HTRF ratios were calculated by dividing the acceptor signal at 665 nm by the donor signal at 620 nm and multiplying by a factor of 10,000. Mean background signal for each target-acceptor concentration (0 nM binder) was subtracted, and data were analyzed using custom Python code by fitting to the general 1 : 1 binding equation where R is the measured equilibrium HTRF signal, A and B are the titrated and fixed binding partner concentrations, respectively, and Rmax and KD are the fitted maximal HTRF signal and binding dissociation constants, respectively. This equation was used because some of the binders had KD values close to or lower than the fixed binder concentration used in the experiment, which causes the more common hyperbolic equation of 1 : 1 binding to overestimate the true KD. TO ensure reliable model fitting, a fixed binder concentration no more than 2-fold higher (and usually much lower) than the estimated KD was always used. Atorney Docket No. 45288-0513WO1

[0531] For selected controls and designs, KDS were measured by kinetic BLI assays to establish confidence in the HTRF results. Data were collected on the Octet R8 (Sartorius AG, Goettingen. Germany) using the integrated Octet Discovery software version 12.2.2.20. Recombinant proteins were diluted from concentrated frozen stocks in 20 mM sodium phosphate pH 7.5, 0.05% Tween-20 (BLI buffer). A seven-point dilution series of the analyte protein was also prepared in BLI buffer to create a titration curve. Ni-NTA biosensors (Sartorius, catalog number 18-5102) were preequilibrated in BLI buffer for at least 10 minutes prior to starting the experiment. A fixed concentration of "ligand" (8His-tagged binder) was loaded onto sensors for 120-240 seconds, briefly washed for 10 seconds, followed by a 60 second baseline. Association of a titration series of "analyte" (target protein) was then performed for 90-420 seconds, followed by dissociation for 600-1200 seconds. All steps were performed at 25 °C and with shaking at 1000 rpm. Loading, association, and dissociation durations were optimized for each binder-target pair. Data were processed using Octet Analysis Studio (version 12.2.2.26). Measurements from reference sensors not loaded with ligand, as well as a reference well with 0 nM analyte, were subtracted from the final data to account for non-specific binding of analyte to the sensors and baseline drift due to unloading of ligand from sensors, respectively. Baseline (pre-association) signal was aligned to 0 before final analysis, where kinetic constants were obtained by nonlinear regression of 1: 1 or 2: 1 binding equations to the data. Fits were performed globally, over both association and dissociation, with a shared Rmax for all analyte concentrations.

[0532] Data were collected on a Jasco J-815 circular dichroism spectrometer, running softw are Spectra Manager Version 2. 15.20. equipped with a PTC-348 temperature control device. Far- UV spectra (260-190 nm) and thermal unfolding measurements w ere recorded in 1 mm quartz glass cuvettes (Hellma) containing protein solutions at 10 pM in 20 mM sodium phosphate pH 7.5.

[0533] Baselines containing 20 mM sodium phosphate pH 7.5 were collected prior to sample analysis. Spectra were recorded in the far-UV (260-190 nm) at 20 °C with a scanning speed of 200 nm / min and a digital integration time (DIT) of 0.25 seconds. 25 accumulations (spectral scans) were recorded and automatically averaged by the software. Thermal unfolding data were recorded at 222 nm between 2-95 °C at a ramp rate of 2 °C / min, with measurements recorded at 0.2 °C intervals. The DIT was set to 4 seconds. Following thermal unfolding measurements, spectra in the far-UV were collected at 95 °C to measure CD spectra changes post thermal Atorney Docket No. 45288-0513WO1 unfolding. Additional CD spectra were then collected following re-cooling of the same samples to 20 °C, to observe refolding.

[0534] Western blot analysis ofVEGF-A signaling in HUVECs

[0535] Human umbilical vein endothelial cells (HUVECs) were thawed from frozen stocks, cultured in media and passaged twice over 7 days before seeding in 6-well non-coated plates.

[0536] 24 hours after seeding, cells were starved for 3 hours in ECG medium with no bullet kit. Following starvation, cells were treated with either no inhibitor, or with VEGFR2 inhibitor control ki8751 (1 pM), anti-VEGF-A inhibitor control Bevacizumab (1 pM), or binders GDM_VEGFA_54 and GDM_VEGFA_71 (1 pM) for 60 minutes by adding each treatment directly into the starvation medium. Following treatment, cells were stimulated with 30 ng / mL hVEGF-A for 2, 5, 10, 30 or 60 minutes (0 minute cells were not treated with hVEGF-A). Following each timepoint, cells were washed with 3 mL ice cold PBS on ice, and care was taken to remove as much PBS as possible before the plates were frozen at -80 °C. Frozen cells were lysed on ice with 60 pL / well ice cold D0.4 lysis buffer (20 mM HEPES pH 7.5, 0.4 M NaCl, 10% glycerol. 0.4% Triton X-100, 10 mM EGTA and 5 mM EDTA) supplemented with lx protease inhibitor cocktail, lx phosphatase inhibitor cocktail, 1 mM DTT, 25 mM NaF and

[0537] 25 mM sodium-b-glycerophosphate. Cells were scraped to collect the lysate, and lysates were centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatants were transferred to fresh 1.5 mL tubes and pellets were discarded. Total protein concentration was determined using the Pierce™ BCA assay (Thermo Fisher Scientific) and lysates were frozen at -80 °C until required for western blots. 15 pL lysates were loaded and proteins were resolved on precast 15-well 4- 12% Bis-Tris SDS-PAGE gels.

[0538] X-rav crystallography sample preparation, data processing and structure solving

[0539] VEGF target and VEGF binder were combined in a ratio of 1 :2.5 in 20 mM sodium phosphate pH 7.5 and incubated at room temperature for Ih with shaking at 1000 rpm. The complex was purified by SEC using a Supderdex 200 increase 10 / 300 gl column (Cytiva), equilibrated with 20 mM Tris pH 7.5, 150 mM NaCl, and verified by SDS-PAGE. The GDM_VEGFA_71 / VEGF-A complex was concentrated to 12 mg / mL in 20 mM Tris pH 7.5 and 150 mM NaCl using a 10 kDa MWCO concentrator (Vivaspin). Cry stallisation was carried out using a Mosquito crystallization robot (SPT Labtech) by sitting-drop vapor diffusion. Crystals of the protein complex grew within two weeks at 20 °C in mother liquor containing 0.1 M phosphate / citrate pH 4.2 and 40 % v / v Ethanol. Atorney Docket No. 45288-0513WO1

[0540] Crystals were harvested with 10 gm Micromounts loops (MiTeGen) and snap frozen in liquid nitrogen prior to data collection. X-ray data were collected from a single crystal at 100 K on the 104 beamline at Diamond Light Source (Harwell, UK) with a wavelength of 0.9537 A.

[0541] All data were automatically processed by xia2. Initial phases for the protein VEGFA- 71 protein complex were obtained by maximum-likelihood molecular replacement using Phaser (version 2.8.3) from the CCP4 Suite (version 9.0.002) with the AF3-predicted structure as search model. The structure solution was subjected to repetitive rounds of restrained refinement using Refmac5 (version 5.8.0430) and interactive manual building in COOT (version 0.9.8.95)7. NCS and Jelly Body restraints were also used throughout the refinement. The final structure quality at 2.56 A was assessed using Molprobity (version 0.9.8.95).

[0542] Example 1 - Protein Design System

[0543] The proteins described herein were designed on a system comprising two components: 1) a "generator", or an all-atom diffusion-based generative model trained on protein structures and sequences from the Protein Data Bank (PDB) as well as a distillation set of AlphaFold predictions; and 2) a "filter" which scores generated designs to predict which are the most likely to succeed experimentally. To design binders, a structure of the "target" protein (e.g., VEGF- A) is input into the system and "hotspot" residues representing the target epitope are optionally designated, and the system then outputs a structure and sequence of a candidate binder for that target. A large number of designs are generated in silico and they are then filtered to a smaller set prior to experimental testing.

[0544] Example 2 - Picomolar-affinity binders from medium-throughput screening

[0545] Binders were designed against a VEGF-A target protein with diverse structural properties (FIG. 1, Table 1).

[0546] Vascular Endothelial Growth Factor A (VEGF-A) is a secreted growth factor controlling angiogenesis and a therapeutic target for cancer and diabetic retinopathy, wherein a small hydrophobic patch bound by the native VEGF receptor was targeted. No designed binders to this target have been published despite its biomedical importance. Atorney Docket No. 45288-0513WO1

[0547] [Table 1] - Binder design problem specifications for in silico benchmarking and experimental testing

[0548] Example 3 - Multiple binding hits within one 96-well plate of designs per target

[0549] For the VEGF-A target, a large set of in silico designs 50-140 amino acids long was generated (Table 1) and a standardized filtering procedure was used to choose between 47 and 94 binder candidates to test for binding by yeast surface display. The protein designs were tested for the VEGF-A target protein, with the experimental success rate ranging from 9.6% to 98%.

[0550] Example 4 - State-of-the-art binding affinities on VEGF-A

[0551] High experimental success rates can reduce the labor and cost of obtaining binders, but once hits have been found, a far more important metric is binding affinity (KD) to the target. Most therapeutic antibodies have low-picomolar KDS, which is achieved by many rounds of experimental optimization. For binders used as research tools, low-nanomolar KD values or better is also typical. To measure how strongly the designed binders bound the VEGF-A target, yeast screening hits were recombinantly expressed and purified in E. coli to measure their KD values in vitro. Overall, 93% of designs chosen for follow up successfully expressed in E. coli (Table 2), and the majority' were monodisperse by size-exclusion chromatography. A subset of designs assayed by circular dichroism (CD) spectroscopy all exhibited high thermostability (Tm > 95°C) and had the expected secondary structures (FIGs. 2D. 5A-5B). For the recombinantly produced designs, KD values were measured using a homogeneous time- resolved fluorescence (HTRF) equilibrium saturation binding assay (FIGs. 2A-2C).

[0552] [Table 2] - Number of yeast hits successfully expressed in E. coli and tested for HTRF binding Atorney Docket No. 45288-0513WO1

[0553] Two binders with picomolar KDS were identified for VEGF-A (FIGs. 6A-6B). Taken together, the success rates and affinities achieved suggest that it can generate binders for many potential applications using only one round of medium-throughput (such as one 96-well plate) screening and no further optimization.

[0554] Example 5 - Designs bind the intended epitope via the intended interactions

[0555] To test whether the designs bind the intended epitope on the target, binding was measured in the presence of a known competitive binder with the same target site. As expected, this reduced binding signal, with the reduction being smaller where the identified VEGF-A binders had a much higher affinity than the competitor. To test whether the VEGF-A binder designs bind their targets via the intended interactions, binding of our top binders w as measured after mutating 1-3 residues at the target-binding interface in their design models (FIGs. 8A- 8B). Almost all mutants had lower binding than their parent, suggesting successful disruption of the binding interface by the mutations. A small number of mutants had higher binding than the parent, but this can likely be explained by idiosyncratic structural factors (FIG. 7). Overall, these results indicate that both the binder and target interact with each other via the interfaces that were intended by design.

[0556] Example 6 - Binders are specific to their targets and are structurally diverse

[0557] Many practical applications require high target specificity, or absence of binding to unintended targets. To test the specificity of a subset of the identified VEGF-A binders, their binding w as measured against all the targets. All binders tested exhibit observable binding only to the intended target (FIG. 9).

[0558] Next, the structural diversity of the binder designs was analyzed to gain insight into how many independent solutions the method is able to generate for each design problem. Diversity7is also practically important as it maximizes the chance that one of the designs will satisfy downstream requirements that are not known in advance. The distribution of pairw ise TM-scores and secondary structure content was observed across binding hits for the VEGF-A target. Compared to the active designs from RF diffusion, the proteins described herein were consistently lower in structural similarity to each other and had a higher frequency of all-beta structures.

[0559] Example 7 - Binders inhibit VEGFR downstream signaling in cells Atorney Docket No. 45288-0513WO1

[0560] The ability to bind target proteins has direct application in biological experimentation. Because the identified VEGF-A binders were designed to bind to target epitopes that normally interact with other proteins to perform biological functions, it was expected that the binders would inhibit these natural functions. To demonstrate this, the abi li ty of the identified VEGF- A binders to inhibit VEGF signaling in human cells by blocking the action of VEGF-A was tested.

[0561] Phosphorylation of VEGF Receptor 2 (VEGFR2) and downstream ERK and AKT kinases were measured in primary human umbilical vein endothelial cells (HUVECs) treated with each of the designed binders GDM_VEGFA_54 or GDM_VEGFA_71 and stimulated with human VEGF-A (FIGs. 3A-3B, 10). Incubation with either binder leads to reduced phosphorylation of ERK and AKT compared to a VEGF-A-only control (FIG. 3 A, "no inhibitor"). Furthermore, GDM VEGFA 54 has a similarly strong effect as ki8751, a smallmolecule VEGFR2 kinase inhibitor, and a stronger effect than Bevacizumab, a clinical mAb binder of VEGF-A that inhibits VEGF-A binding to VEGFR2.

[0562] Example 8 - Experimental structures of binder-target complexes confirm binding mode and structure

[0563] To validate the structures and binding modes of the identified binder designs, an x-ray cry stal structure of GDM_VEGFA_71 was obtained in complex with VEGF-A, at 2.65 A resolution (FIG. 4A). The binder folded to its designed structure, a mixed alpha-beta fold with a 5-strand beta sheet interacting with VEGF-A, with atomic accuracy, with a Ca RMSD of 0.78 A between AF3 model and experimental structure (FIG. 4B). The designed binding site was also highly accurate, with a target-aligned binder Ca RMSD of 1.65 A.

[0564] Most sidechains of the binder interacting with the target also had the correct rotamer, including a buried hydrogen bond between a histidine of the binder and a tyrosine of VEGF-A which was recapitulated almost perfectly in the experimental structure (FIGs. 4C-4D).

[0565] B. Epstein-Barr Virus BCL-2 homolog (BHRF1) protein

[0566] Methods

[0567] For BHRF1, a recombinant protein construct (Uniprot P03182, residues 2-160) was produced with an N-terminal Twin-Strep tag and a 3C protease cleavage site. BL21 (DE3) cultures were grown in Terrific Broth (TB) medium (Melford) supplemented with carbenicillin Atorney Docket No. 45288-0513WO1

[0568] (50 pg / mL) at 37°C with shaking. At OD600 = 0.6, protein expression was induced with 0.1 mM IPTG, the temperature was reduced to 21 °C and cultures were grown overnight.

[0569] Cells were harvested and resuspended in 20 mM Tris pH 8.0, 300 mM NaCl supplemented with 0.5 mg / mL lysozyme, 100 U DNAse I, 1 mM MgC12 and a cOmplete EDTA-free protease inhibitor tablet (Roche) at a 1 :5 cell weight to buffer ratio. Cell lysis was achieved by sonicating the cell suspension at 40% amplitude (15 sec on / 45 sec off) for 24 cycles on ice. Lysate was centrifuged at 48,000 x g for 45 min at 4 °C and the supernatant was recovered and filtered through a 0.45 pm filter (Sartorius). The sample was applied to a 5 mL StrepTrap XT column (Cytiva) pre-equilibrated with Strep binding buffer (100 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM TCEP) using an AKTA Pure 25 M. Following sample application, the column resin was washed with 10 column volumes (CV) of the same buffer before the protein was eluted with 10 CV of lx BXT elution buffer (IB A Lifesciences) supplemented with 0.5 mM TCEP. 1 CV fractions were collected and assessed via SDS-PAGE to confirm presence of the protein of interest. BHRF1 was pooled and concentrated using a 10,000 kDa MWCO concentrator (Vivaspin). The sample was further purified by SEC using a Superdex 75 increase 10 / 300 GL column pre-equilibrated with 20 mM sodium phosphate pH 7.5, 0.5 mM TCEP. Fractions were confirmed by SDS-PAGE and the concentration was measured by absorbance at 280 nm using a NanoDrop One (Thermo Scientific) and the BHRF1 construct’s theoretical extinction coefficient. Purified protein was aliquoted and stored at -80 °C.

[0570] Yeast surface display and flow cytometry

[0571] Primary binding screen

[0572] Binder design sequences were codon-optimized by DNAworks and most were synthesized by Twist as gene fragments flanked by Bsal restriction sites as well as homology regions to a modified pETcon vector. Saccharomyces cerevisiae strain EBY100 cells (50 pL) were transformed using the lithium acetate method with 50 ng of linearized plasmid and a minimum of 10 ng of gene fragment insert in a 96-well plate. Cells were grown at 30 °C shaking at 1,000 rpm in complete synthetic medium -Trp -Ura + 2% glucose for 48-72 h. For protein expression, 2 x 107yeast cells were centrifuged at 1,800 x g for 5 mins at 20 °C and resuspended in 1 mL complete synthetic medium + 0.1% glucose + 2% galactose (SGCAA). Cells were incubated at 30 °C overnight (16-18 hours) and 8 x 106yeast cells were washed twice with 200 pL of lx PBS + 0.1% BSA (PBSF). centrifuged at 1,800 x g for 3 min at 20 °C and the supernatant was removed. Atorney Docket No. 45288-0513WO1

[0573] To screen for binding, yeast cells were then incubated with biotinylated target proteins (diluted in PBSF) for 1 hour, washed twice with PBSF and incubated with 25 ug / mL fluorescein isothiocyanate (FITC)-conjugated anti-Myc antibody (FITC-Ab) (Abeam) and 30 ug / mL streptavidin-phycoerythrin (SAPE, Thermo Fisher Scientific) for 30 minutes. Following binding, cells were w ashed once with PBSF and resuspended in 200 pL of PBSF. Cells were analyzed on the CytoFlex LX (Beckman Coulter) or ZE5 Cell Analyzer (Bio-Rad) flow cytometers by measuring fluorescence of FITC and phycoerythrin (PE) to detect binder expression and target binding respectively.

[0574] Interface mutation, competitive inhibition, and specificity experiments

[0575] Mutants were screened following the same method as the primary binding screen. For competition assays, yeast cells were incubated with biotinylated target proteins with or without a competitor protein for 1 h (the competitor protein was added to the biotinylated target protein master mix just before pipetting to the cells). The cells w ere then washed twice with PBSF and incubated with 25 pg / mL FITC-Ab (Abeam) and 30 pg / mL SAPE (Thermo Fisher Scientific) for 30 minutes. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. To test for specificity, specific designs as well as pooled cells were tested against target protein following the same method as the primary binding screen.

[0576] Designed binder expression and purification

[0577] Designed binders with the highest binding signal by yeast display were selected for E. coli expression and follow^ up experiments. Designs purchased as gene fragments were cloned into a modified pTriEx-4 vector containing an N-terminal 8-His tag using NEBridge Golden Gate cloning (NEB) at Bsal sites, transformed into DH5-a competent cells, miniprepped (Qiagen), and verified by Sanger sequencing (Azenta). A small number of designs were purchased from Twist Bioscience directly as cloned plasmids in pTriEx-4 or pET-29b. For expression, plasmids were transformed into BL21 (DE3) cells and the entire transformation mix inoculated into autoinduction medium consisting of Terrific Broth (TB) medium (Melford). 0.05% glucose, 0.2% alpha-lactose, and 50 p / mL carbenicillin. Cultures were incubated at 37 °C with shaking (220 or 1000 rpm) for 24 hours, harvested at 2,568 x g for 10 minutes, and pellets stored at -80 °C until purification. Cell pellets were lysed by chemical lysis using BugBuster Master Mix (Novagen) supplemented with cOmplete EDTA-free protease inhibitor (Roche) with shaking for 20 minutes at room temperature. Lysates were clarified by centrifugation for 1 hour at 2,568 x g, then purified by immobilized metal affinity Atorney Docket No. 45288-0513WO1 chromatography (IMAC) using Ni-NTA in either 0. 1 mL spin columns (Cytiva) or in HisPur™ Ni-NTA 96-well Spin Plates (Thermo Scientific), followed by size exclusion chromatography (SEC) on an AKTA Pure 25 M (Cytiva) equipped with an ALIAS autosampler (Spark Holland) using a Superdex 75 increase 10 / 300 GL column equilibrated in 20 mM sodium phosphate pH 7.5. Protein samples were analyzed by SDS-PAGE, and where required, concentrated using a 3 kDa MWCO Vivaspin concentrator (Cytiva). Protein concentrations were measured in triplicate by absorbance at 280 nm with a NanoDrop One (Thermo Scientific) using theoretical extinction coefficients, or using custom Python code. Purified proteins were aliquoted and stored at -80 pC until further use.

[0578] Where larger quantities of designed binders were required, for example for circular dichroism and x-ray crystallography experiments, expression was scaled up to 100-1000 mL BL21 (DE3) cultures and the above protocol followed with minor modifications. Cells were lysed using sonication for a total of 6 minutes (5 sec on / 5 sec off) on ice, and lysates were clarified by centrifugation at 40,000 x g for 45 minutes before being applied to 5 mL Ni-NTA column (Cytiva). followed by SEC.

[0579] Homogeneous Time Resolved Fluorescence (HTRF)

[0580] Measurement of binding affinity binding dissociation constants (KD)

[0581] Binding affinities (KDS) were measured in equilibrium saturation-binding experiments with fixed binder design concentration and target titration. The total assay volume was 16 pL and all proteins and reagents were diluted in PPI europium detection buffer (Revvity). Target protein was premixed with HTRF acceptor reagent Streptavidin-d2 (Revvity), serially diluted, and transferred to a white ProxiPlate 384-shallow well microplate (‘assay plate’, Revvity). Subsequently, 1 nM of each binder was added to the assay plate in duplicate (binders with KD < 0.5 nM were later re-assayed with 0.1 nM binder to ensure robust data fitting). The assay plate was centrifuged at 500 x g for 30 seconds, sealed and incubated at room temperature for between 30 minutes and 1 hour. HTRF donor mAb Anti-6HIS-Eu Gold (Revvity) was then added to a final concentration of 2 nM (lx), using a Mantis microfluidic liquid dispenser (Formulatrix) running software version 5. 1. 1 on Windows 10. The assay plate was centrifuged, sealed and incubated for a further 1 hour at room temperature.

[0582] HTRF signal was measured using a PHERAstar FSX (BMG) plate reader equipped with an HTRF 337 / 665 / 620 optic module running software version 5.70 R6 on Windows 10. The measurement conditions were as follows; 60 ps integration delay, 400 ps integration time, 60 flashes. The optimal focal (Z) height was determined using channel B for each experiment. Atorney Docket No. 45288-0513WO1

[0583] HTRF ratios were calculated by dividing the acceptor signal at 665 nm by the donor signal at 620 nm and multiplying by a factor of 10,000. Mean background signal for each target-acceptor concentration (0 nM binder) was subtracted, and data were analyzed using custom Python code by fitting to the general 1 : 1 binding equation where R is the measured equilibrium HTRF signal, A and B are the titrated and fixed binding partner concentrations, respectively, and Rmax and KD are the fitted maximal HTRF signal and binding dissociation constants, respectively. This equation was used because some of the binders had KD values close to or lower than the fixed binder concentration used in the experiment, which causes the more common hyperbolic equation of 1 : 1 binding to overestimate the true KD. TO ensure reliable model fitting, a fixed binder concentration no more than 2-fold higher (and usually much lower) than the estimated KD was always used.

[0584] For selected controls and designs, KDS were measured by kinetic BLI assays to establish confidence in the HTRF results. Data were collected on the Octet R8 (Sartorius AG, Goettingen. Germany) using the integrated Octet Discovery software version 12.2.2.20. Recombinant proteins were diluted from concentrated frozen stocks in 20 rnM sodium phosphate pH 7.5, 0.05% Tween-20 (BLI buffer). A seven-point dilution series of the analyte protein was also prepared in BLI buffer to create a titration curve. Ni-NTA biosensors (Sartorius, catalog number 18-5102) were preequilibrated in BLI buffer for at least 10 minutes prior to starting the experiment. A fixed concentration of "ligand" (8His-tagged binder) was loaded onto sensors for 120-240 seconds, briefly washed for 10 seconds, followed by a 60 second baseline. Association of a titration series of "analyte" (target protein) was then performed for 90-420 seconds, followed by dissociation for 600-1200 seconds. All steps were performed at 25 °C and with shaking at 1000 rpm. Loading, association, and dissociation durations were optimized for each binder-target pair. Data were processed using Octet Analysis Studio (version 12.2.2.26). Measurements from reference sensors not loaded with ligand, as w ell as a reference well with 0 nM analyte, were subtracted from the final data to account for non-specific binding of analyte to the sensors and baseline drift due to unloading of ligand from sensors, respectively. Baseline (pre-association) signal w as aligned to 0 before final analysis, where kinetic constants were obtained by nonlinear regression of 1: 1 or 2: 1 binding equations to the data. Fits were Atorney Docket No. 45288-0513WO1 performed globally, over both association and dissociation, with a shared Rmax for all analyte concentrations.

[0585] Data were collected on a Jasco J-815 circular dichroism spectrometer, running software Spectra Manager Version 2.15.20, equipped with a PTC-348 temperature control device. Far- UV spectra (260-190 nm) and thermal unfolding measurements were recorded in 1 mm quartz glass cuvettes (Hellma) containing protein solutions at 10 pM in 20 mM sodium phosphate pH 7.5.

[0586] Baselines containing 20 mM sodium phosphate pH 7.5 were collected prior to sample analysis. Spectra were recorded in the far-UV (260-190 nm) at 20 °C with a scanning speed of 200 nm / min and a digital integration time (DIT) of 0.25 seconds. 25 accumulations (spectral scans) were recorded and automatically averaged by the software. Thermal unfolding data were recorded at 222 nm between 2-95 °C at a ramp rate of 2 °C / min, with measurements recorded at 0.2 °C intervals. The DIT was set to 4 seconds. Following thermal unfolding measurements, spectra in the far-UV were collected at 95 °C to measure CD spectra changes post thermal unfolding. Additional CD spectra were then collected following re-cooling of the same samples to 20 °C, to observe refolding.

[0587] Example 9 - Protein Design System

[0588] The proteins described herein were designed on a system comprising two components: 1) a "generator", or an all-atom diffusion-based generative model trained on protein structures and sequences from the Protein Data Bank (PDB) as well as a distillation set of AlphaFold predictions; and 2) a "filter" which scores generated designs to predict which are the most likely to succeed experimentally. To design binders, a structure of the "target" protein (e.g., BHRF1) is input into the system and "hotspot" residues representing the target epitope are optionally designated, and the system then outputs a structure and sequence of a candidate binder for that target. A large number of designs are generated in silico and they are then filtered to a smaller set prior to experimental testing.

[0589] Example 10 - Picomolar-affinity binders from medium-throughput screening

[0590] Binders were designed against a BHRF1 target protein with diverse structural properties (FIG. 11, Table 3). Atorney Docket No. 45288-0513WO1

[0591] BHRF1 is an oncogenic protein from Epstein-Barr virus; inhibiting it via binding can kill cancer cells and slow tumor growth. It has a hydrophobic groove that perfectly accommodates a helix on its binding partner, making it a relatively easy problem.

[0592] [Table 3] - Binder design problem specifications for in silico benchmarking and experimental testing

[0593] Example 11 - Multiple binding hits within one 96-well plate of designs per target

[0594] For the BHRF1 target, a large set of in silico designs 50-140 amino acids long was generated (Table 3) and a standardized filtering procedure was used to choose between 47 and 94 binder candidates to test for binding by yeast surface display. The designs were tested for the BHRF1 target protein, with the experimental success rate ranging from 9.6% to 98%.

[0595] Example 12 - State-of-the-art binding affinities on BHRF1

[0596] High experimental success rates can reduce the labor and cost of obtaining binders, but once hits have been found, a far more important metric is binding affinity (KD) to the target. Most therapeutic antibodies have low-picomolar KDS, which is achieved by many rounds of experimental optimization. For binders used as research tools, low-nanomolar KD values or better is also ty pical. To measure how strongly the designed binders bound the BHRF1 target, yeast screening hits were recombinantly expressed and purified in E. coli to measure their KD values in vitro. Overall, 93% of designs chosen for follow up successfully expressed in E. coli (Table 4), and the majority7were monodisperse by size-exclusion chromatography. A subset of designs assayed by circular dichroism (CD) spectroscopy all exhibited high thermostability (Tm > 95°C) and had the expected secondary' structures. For the recombinantly produced designs, KD values were measured using a homogeneous time-resolved fluorescence (HTRF) equilibrium saturation binding assay (FIGs. 12A-12G).

[0597] [Table 4] - Number of yeast hits successfully expressed in E. coli and tested for HTRF binding Atorney Docket No. 45288-0513WO1

[0598] The KDS of the presently described proteins were better on the BHRF1 target protein, by a margin of 1.75-for BHRF1. Even compared to previous designed binders that have been optimized experimentally through multiple rounds of mutation and selection, the best KD values were still better on BHRF1. Taken together, the success rates and affinities achieved by the present proteins suggest that this generator can provide binders for many potential applications using only one round of medium-throughput (such as one 96-well plate) screening and no further optimization (FIGs. 13A-13B).

[0599] Example 13 - Designs bind the intended epitope via the intended interactions

[0600] To test whether the designs bind the intended epitope on the target, binding was measured in the presence of a known competitive binder with the same target site. As expected, this reduced binding signal, with the reduction being smaller where the identified BHRF1 binders had a much higher affinity than the competitor. To test whether the BHRF1 binder designs bind their targets via the intended interactions, we measured binding of our top binders was measured after mutating 1-3 residues at the target-binding interface in their design models (FIGs. 15A-15B). Almost all mutants had lower binding than their parent, suggesting successful disruption of the binding interface by the mutations. A small number of mutants had higher binding than the parent, but this can likely be explained by idiosyncratic structural factors (FIG. 14) Overall, these results indicate that both the binder and target interact with each other via the interfaces that were intended by design.

[0601] Example 14 - Binders are specific to their targets and are structurally diverse

[0602] Many practical applications require high target specificity, or absence of binding to unintended targets. To test the specificity of a subset of the identified BHRF1 binders, their binding was measured against all the targets. All binders tested exhibit observable binding only to the intended target (FIG. 9).

[0603] Next, the structural diversity of the binder designs was analyzed to gain insight into how many independent solutions the method is able to generate for each design problem. Diversity is also practically important as it maximizes the chance that one of the designs will satisfy ownstream requirements that are not known in advance. The distribution of painvise Atorney Docket No. 45288-0513WO1

[0604] TM-scores and secondary structure content was observed across binding hits for the BHRF1 target. Compared to the active designs from RF diffusion, the proteins described in the present disclosure were consistently lower in structural similarity to each other and had a higher frequency of all-beta structures.

[0605] C. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) Receptor Binding Domain (RBD)

[0606] Methods

[0607] Target protein expression and purification

[0608] For SC2RBD, a recombinant protein construct (NCBI reference NC_045512, residues 319-541) of SARS-CoV 2 Spike S 1 glycoprotein corresponding to the receptor binding domain was produced with a C-terminal Twin-Strep tag. The signal peptide from immunoglobulin kappa gene product (METDTLLLWVLLLWVPGSTGD; SEQ ID NO: 33) was used to direct secretion of the construct. The corresponding codon-optimised DNA fragment was cloned into mammalian expression vector pQ-3C-2xStrep for expression in human Expi293F cells. Expi293F cells grown at 37 °C in 5% CO2 in shake flasks containing FreeStyle 293 medium were transfected with endotoxin free plasmid preparation using ExpiFectamine reagent (Thermo Fisher Scientific).

[0609] Recombinant protein was captured on Streptactin XT (IBA LifeSciences) affinity resin. Following extensive washes in TBSE buffer (20 mM Tris-HCl pH 8.0. 150 mM NaCl, 1 mM EDTA), the protein was eluted in lx BXT buffer (IBA LifeSciences) and further purified by size exclusion chromatography using a Superdex 200 16 / 600 column (GE Healthcare) in TBSE buffer. The purified protein was concentrated using a 10,000 kDa MWCO concentrator (Sartorius), aliquoted, snap-frozen in liquid nitrogen and stored at -80 °C.

[0610] Yeast surface display and flow cytometry

[0611] Primary binding screen

[0612] Binder design sequences were codon-optimized by DNAworks and most were synthesized by Twist as gene fragments flanked by Bsal restriction sites as well as homology regions to a modified pETcon vector. Saccharomyces cerevisiae strain EBY100 cells (50 pL) were transformed using the lithium acetate method with 50 ng of linearized plasmid and a minimum of 10 ng of gene fragment insert in a 96-well plate. Cells were grown at 30 °C shaking at 1,000 rpm in complete synthetic medium -Trp -Ura + 2% glucose for 48-72 h. For protein expression, 2 x 107yeast cells were centrifuged at 1,800 x g for 5 mins at 20 °C and resuspended Atorney Docket No. 45288-0513WO1 in 1 mL complete synthetic medium + 0.1% glucose + 2% galactose (SGCAA). Cells were incubated at 30 °C overnight (16-18 hours) and 8 x 106yeast cells were washed twice with 200 pL of lx PBS + 0.1% BSA (PBSF), centrifuged at 1,800 x g for 3 min at 20 °C and the supernatant was removed.

[0613] To screen for binding, yeast cells were then incubated with biotinylated target proteins (diluted in PBSF) for 1 hour, washed twice with PBSF and incubated with 25 ug / mL fluorescein isothiocyanate (FITC)-conjugated anti-Myc antibody (FITC-Ab) (Abeam) and 30 ug / mL streptavidin-phycoerythrin (SAPE, Thermo Fisher Scientific) for 30 minutes. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. Cells were analyzed on the CytoFlex LX (Beckman Coulter) or ZE5 Cell Analyzer (Bio-Rad) flow cytometers by measuring fluorescence of FITC and phycoerythrin (PE) to detect binder expression and target binding respectively.

[0614] Interface mutation, competitive inhibition, and specificity experiments

[0615] Mutants were screened following the same method as the primary’ binding screen. For competition assays, yeast cells were incubated with biotinylated target proteins with or without a competitor protein for 1 h (the competitor protein was added to the biotinylated target protein master mix just before pipetting to the cells). The cells w ere then w ashed tw ice with PBSF and incubated with 25 pg / mL FITC-Ab (Abeam) and 30 pg / mL SAPE (Thermo Fisher Scientific) for 30 minutes. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. To test for specificity, specific designs as well as pooled cells were tested against target protein following the same method as the primary' binding screen.

[0616] Designed binder expression and purification

[0617] Designed binders with the highest binding signal by yeast display were selected for E. coli expression and follow' up experiments. Designs purchased as gene fragments were cloned into a modified pTriEx-4 vector containing an N-terminal 8-His tag using NEBridge Golden Gate cloning (NEB) at Bsal sites, transformed into DH5-a competent cells, miniprepped (Qiagen), and verified by Sanger sequencing (Azenta). A small number of designs were purchased from Twist Bioscience directly as cloned plasmids in pTriEx-4 or pET-29b. For expression, plasmids w ere transformed into BL21 (DE3) cells and the entire transformation mix inoculated into autoinduction medium consisting of Terrific Broth (TB) medium (Melford). 0.05% glucose, 0.2% alpha-lactose, and 50 p / mL carbenicillin. Cultures were incubated at 37 °C with shaking (220 or 1000 rpm) for 24 hours, harvested at 2,568 x g for 10 minutes, and Atorney Docket No. 45288-0513WO1 pellets stored at -80 °C until purification. Cell pellets were lysed by chemical lysis using BugBuster Master Mix (Novagen) supplemented with complete EDTA-free protease inhibitor (Roche) with shaking for 20 minutes at room temperature. Lysates were clarified by centrifugation for 1 hour at 2,568 x g, then purified by immobilized metal affinity chromatography (IMAC) using Ni-NTA in either 0. 1 mL spin columns (Cytiva) or in HisPur™ Ni-NTA 96-well Spin Plates (Thermo Scientific), followed by size exclusion chromatography (SEC) on an AKTA Pure 25 M (Cytiva) equipped with an ALIAS autosampler (Spark Holland) using a Superdex 75 increase 10 / 300 GL column equilibrated in 20 mM sodium phosphate pH 7.5. Protein samples were analyzed by SDS-PAGE, and where required, concentrated using a 3 kDa MWCO Vivaspin concentrator (Cytiva). Protein concentrations were measured in triplicate by absorbance at 280 nm with a NanoDrop One (Thermo Scientific) using theoretical extinction coefficients, or using custom Python code. Purified proteins were aliquoted and stored at -80 pC until further use.

[0618] Where larger quantities of designed binders were required, for example for circular dichroism and x-ray crystallography experiments, expression was scaled up to 100-1000 mL BL21 (DE3) cultures and the above protocol followed with minor modifications. Cells were lysed using sonication for a total of 6 minutes (5 sec on / 5 sec off) on ice, and lysates were clarified by centrifugation at 40,000 x g for 45 minutes before being applied to 5 mL Ni-NTA column (Cytiva). followed by SEC.

[0619] Homogeneous Time Resolved Fluorescence (HTRF)

[0620] Measurement of binding affinity / binding dissociation constants (KD)

[0621] Binding affinities (KDS) were measured in equilibrium saturation-binding experiments with fixed binder design concentration and target titration. The total assay volume was 16 pL and all proteins and reagents were diluted in PPI europium detection buffer (Revvity). Target protein was premixed with HTRF acceptor reagent Streptavidin-d2 (Revvity), serially diluted, and transferred to a white ProxiPlate 384-shallow well microplate (‘assay plate’, Revvity ). Subsequently, 1 nM of each binder was added to the assay plate in duplicate (binders with KD < 0.5 nM were later re-assayed with 0.1 nM binder to ensure robust data fitting). The assay plate was centrifuged at 500 x g for 30 seconds, sealed and incubated at room temperature for between 30 minutes and 1 hour. HTRF donor mAb Anti-6HIS-Eu Gold (Revvity ) was then added to a final concentration of 2 nM (lx), using a Mantis microfluidic liquid dispenser (Formulatrix) running software version 5.1.1 on Windows 10. The assay plate was centrifuged, sealed and incubated for a further 1 hour at room temperature. Atorney Docket No. 45288-0513WO1

[0622] HTRF signal was measured using a PHERAstar FSX (BMG) plate reader equipped with an HTRF 337 / 665 / 620 optic module running software version 5.70 R6 on Windows 10. The measurement conditions were as follows; 60 ps integration delay, 400 ps integration time, 60 flashes. The optimal focal (Z) height was determined using channel B for each experiment. HTRF ratios were calculated by dividing the acceptor signal at 665 nm by the donor signal at 620 nm and multiplying by a factor of 10,000. Mean background signal for each target-acceptor concentration (0 nM binder) was subtracted, and data were analyzed using custom Python code by fitting to the general 1 : 1 binding equation where R is the measured equilibrium HTRF signal, A and B are the titrated and fixed binding partner concentrations, respectively, and Rmax and KD are the fitted maximal HTRF signal and binding dissociation constants, respectively. This equation was used because some of the binders had KD values close to or lower than the fixed binder concentration used in the experiment, w hich causes the more common hyperbolic equation of 1 : 1 binding to overestimate the true KD. TO ensure reliable model fitting, a fixed binder concentration no more than 2-fold higher (and usually much lower) than the estimated KD was always used.

[0623] Bio-Layer Interferometry (BLI)

[0624] For selected controls and designs, KDS were measured by kinetic BLI assays to establish confidence in the HTRF results. Data were collected on the Octet R8 (Sartorius AG, Goettingen. Germany) using the integrated Octet Discovery software version 12.2.2.20. Recombinant proteins were diluted from concentrated frozen stocks in 20 mM sodium phosphate pH 7.5, 0.05% Tween-20 (BLI buffer). A seven-point dilution series of the analyte protein was also prepared in BLI buffer to create a titration curve. Ni-NTA biosensors (Sartorius, catalog number 18-5102) were preequilibrated in BLI buffer for at least 10 minutes prior to starting the experiment. A fixed concentration of "ligand" (8His-tagged binder) was loaded onto sensors for 120-240 seconds, briefly washed for 10 seconds, follow ed by a 60 second baseline. Association of a titration series of "analyte" (target protein) was then performed for 90-420 seconds, followed by dissociation for 600-1200 seconds. All steps were performed at 25 °C and with shaking at 1000 rpm. Loading, association, and dissociation durations were optimized for each binder-target pair. Data were processed using Octet Analysis Studio (version 12.2.2.26). Measurements from reference sensors not loaded with ligand, as w ell as a reference Atorney Docket No. 45288-0513WO1 well with 0 nM analyte, were subtracted from the final data to account for non-specific binding of analyte to the sensors and baseline drift due to unloading of ligand from sensors, respectively. Baseline (pre-association) signal was aligned to 0 before final analysis, where kinetic constants were obtained by nonlinear regression of 1 : 1 or 2: 1 binding equations to the data. Fits were performed globally, over both association and dissociation, with a shared Rmax for all analyte concentrations.

[0625] Circular dichroism (CD) spectroscopy

[0626] Data were collected on a Jasco J-815 circular dichroism spectrometer, running software Spectra Manager Version 2. 15.20, equipped with a PTC-348 temperature control device. Far- UV spectra (260-190 nm) and thermal unfolding measurements were recorded in 1 mm quartz glass cuvettes (Hellma) containing protein solutions at 10 pM in 20 mM sodium phosphate pH 7.5.

[0627] Baselines containing 20 mM sodium phosphate pH 7.5 were collected prior to sample analysis. Spectra were recorded in the far-UV (260-190 nm) at 20 °C with a scanning speed of 200 nm / min and a digital integration time (DIT) of 0.25 seconds. 25 accumulations (spectral scans) were recorded and automatically averaged by the software. Thermal unfolding data were recorded at 222 nm between 2-95 °C at a ramp rate of 2 °C / min, with measurements recorded at 0.2 °C intervals. The DIT was set to 4 seconds. Following thermal unfolding measurements, spectra in the far-UV were collected at 95 °C to measure CD spectra changes post thermal unfolding. Additional CD spectra were then collected following re-cooling of the same samples to 20 °C, to observe refolding.

[0628] Experiments were performed by the Francis Crick Covid Surveillance Unit. Briefly, 10-point binder dose response series were generated by serially diluting each binder in duplicate in 20 mM sodium phosphate buffer before diluting further to achieve final testing concentrations of 1.7-11,200 ng / mL in 10% fetal bovine serum (FBS). With appropriate positive and negative controls, binder dose response series were then run through the standard live-virus neutralization assay against 2 variants of concern (VOCs) and 2 variants of interest. Duplicate assay plates were run, so each biological repeat contained 4 technical replicates. 2 biological repeats were run on separate days using different flasks of cells, vials of virus, and bottles of media. Thus each plot of FIG. 24 consists of 160 independent data points. The data Atorney Docket No. 45288-0513WO1 points were generated from 4 replicates of 40 independent titrations. EC50 values were calculated using nonlinear regression with a 4-parameter dose response curve fit.

[0629] The Spike ectodomain construct used in the cryo-EM experiments was based on Wuhan SARS-CoV-2 isolate. The protein (spanning residues 1-1208 from UniProt ID YP_009724390) harbored point mutations K986P and V987P stabilizing the pre-fusion conformation, disrupted furin cleavage site, C-terminal T4 fibritin trimerization domain, and a hexa-histidine affinity tag. The protein was produced by expression in a stably transformed human cell line and purified by capture onto immobilized Ni affinity resin, followed by size exclusion chromatography.

[0630] Four pl freshly isolated trimeric SARS-CoV-2 Spike ectodomain (1.2 mg / mL), supplemented with 0.2 mg / mL acro-0654, acro-0882, acro-0514, or acro-0679 and 0.1% n-octyl glucoside in 150 mM NaCl, 20 mM Tris-HCl, pH 8.0, was spotted onto fresh 400-mesh Rl.2 / 1.3 C-flat holey carbon grids (Electron Microscopy Sciences product CF413-50-Au, used without glow discharging) for 1 min, under 100% humidity at 20°C, prior to blotting and plunge-freezing in liquid ethane-propane using Vitrobot Mark IV (Thermo Fisher Scientific). Cryo-EM data were acquired on a Titan Krios G3i cryo-electron microscope equipped with a Falcon 4i direct electron detector (Thermo Fisher Scientific). Selectris energy filter (Thermo Fisher Scientific) with a slit width of 10 eV was used for imaging complexes containing acro-0514 and acro- 0679. A total of 4500, 8342, 6728, and 8482 micrograph movies were recorded from grids containing acro-0654, acro-0882, acro-0514, and acro-0679, respectively. Data collections proceeded with a defocus range set to -1.5 to -3.5 pm and a magnification corresponding to calibrated pixel size of 1.08 A (acro-0654 and acro-0882) or 0.95 A (acro-0514 and acro-0679).

[0631] 1,674 EER frames recorded per micrograph movie were processed into 31 fractions, with an exposure dose of 1.04 e / A2 (acro-0654 and acro-0882) or 1.25 e / A2 (acro-0514 and acro-0679) per fraction. The micrograph movies were aligned, summed and weighted as implemented in Relion-5. Obeta, and contrast transfer function parameters were estimated using Gctf-vl.18. Reference-free 2D classification of an initial subset of particles picked using gaussian blob function in Relion revealed 2D averages belonging to monomeric SI protein, presumably due to dissociation of the trimeric Spike. Particles belonging to w ell-defined 2D classes were used to train Topaz, which w as used to pick the images. The particles, extracted with 4-fold binning, were subjected to three rounds of 2D classification in Relion, using 400 classes in each round; the regularization parameter T was increased from 2 during the first Atorney Docket No. 45288-0513WO1 round to 8 in the last round of 2D classification. Particles contributing to well-defined 2D classes, re-extracted with 2-fold binning, were used to generate initial 3D models and subjected to 3D classification into 4-7 classes in Relion, with the regularization parameter T set to 8 (Table 5). The best particle sets were used for 3D reconstruction, followed by Bayesian polishing. The final reconstructions were obtained using soft masks in conjunction with Blush regularization, as implemented in Relion-5. Obeta. Resolution metrics reported in this work were according to the gold-standard Fourier shell correlation (FSC) 0.143 criterion. For illustration purposes, cryo-EM maps were locally filtered using EMReady. Rigid body docking of SI protein chain (from PDB ID 7ZBU) and binder models into the final cryo- EM maps was done, and the figures were prepared using PyMOL Molecular Graphics System, Version 3.0 (Schrodinger, LLC).

[0632] Example 15 - Protein Design System

[0633] The proteins described herein were designed on a system comprising two components: 1) a "generator", or an all-atom diffusion-based generative model trained on protein structures and sequences from the Protein Data Bank (PDB) as well as a distillation set of AlphaFold predictions; and 2) a "filter" which scores generated designs to predict which are the most likely to succeed experimentally. To design binders, a structure of the "target" protein (e.g., SARS- CoV-2 RBD) is input into the system and "hotspot" residues representing the target epitope are optionally designated, and the system then outputs a structure and sequence of a candidate binder for that target. A large number of designs are generated in silico and they are then filtered to a smaller set prior to experimental testing.

[0634] Example 16 - Picomolar-affinity binders from medium-throughput screening

[0635] Binders were designed against a SARS-CoV-2 RBD target protein with diverse structural properties (FIG. 16, Table 5).

[0636] SARS-CoV-2 spike protein receptor-binding domain (SC2RBD) is a protein domain required for COVID- 19 infection. The protein interface to the human ACE2 receptor was targeted as disrupting this interaction is known to block SARS-CoV-2 from infecting human cells. Previous design efforts have succeeded against this polar and convex site but required experimental optimization to achieve high affinity . Atorney Docket No. 45288-0513WO1

[0637] [Table 5] - Binder design problem specifications for in silico benchmarking and experimental testing

[0638] Example 17 - Multiple binding hits within one 96-well plate of designs per target

[0639] For the SARS-CoV-2 RBD target, a large set of in silico designs 50-140 amino acids long was generated (Table 5) and a standardized filtering procedure was used to choose between 47 and 94 binder candidates to test for binding by yeast surface display. Designs were tested for the SARS-CoV-2 RBD target protein, with the experimental success rate ranging from 9.6% to 98%. For SC2RBD, additional rounds of up to 31 designs were tested with improved in silico filters or using the B2 system. These follow up rounds generally saw higher success rates, for a maximum per-round success rate of 29% on SC2RBD.

[0640] Example 18 - State-of-the-art binding affinities on SARS-CoV-2 RBD

[0641] High experimental success rates can reduce the labor and cost of obtaining binders, but once hits have been found, a far more important metric is binding affinity (KD) to the target. Most therapeutic antibodies have low-picomolar KDS, which is achieved by many rounds of experimental optimization. For binders used as research tools, low-nanomolar KD values or better is also typical. To measure how strongly the designed binders bound the SARS-CoV-2 RBD target, yeast screening hits were recombinantly expressed and purified in E. coli to measure their KD values in vitro. Overall, 93% of designs chosen for follow up successfully expressed in E. coli (Table 6), and the majority were monodisperse by size-exclusion chromatography. A subset of designs assayed by circular dichroism (CD) spectroscopy all exhibited high thermostability (Tm > 95°C) and had the expected secondary structures (FIGs. 17D, 17F, 20A-20C). For the recombinantly produced designs, KD values were measured using a homogeneous time-resolved fluorescence (HTRF) equilibrium saturation binding assay (FIGs. 17A-17C, 17E)

[0642] [Table 6] - Number of yeast hits successfully expressed in E. coli and tested for HTRF binding Atorney Docket No. 45288-0513WO1

[0643] Compared to the best unoptimized binders from other design methods, the present proteins exhibited better KDS by margins of 4-fold for SC2RBD. Taken together, the success rates and affinities described herein suggest that the design system binders for many potential applications using only one round of medium-throughput (such as one 96-well plate) screening and no further optimization (FIGs. 21A-21B).

[0644] Example 19 - Designs bind the intended epitope via the intended interactions

[0645] To test whether the designs bind the intended epitope on the target, binding was measured in the presence of a know n competitive binder with the same target site. As expected, this reduced binding signal, with the reduction being smaller where the identified SARS-CoV- 2 RBD binders had a much higher affinity than the competitor. To test whether the SARS- CoV-2 RBD binder designs bind their targets via the intended interactions, binding of our top binders was measured after mutating 1-3 residues at the target-binding interface in their design models (FIGs. 23A-23C). Almost all mutants had lower binding than their parent, suggesting successful disruption of the binding interface by the mutations. A small number of mutants had higher binding than the parent, but this can likely be explained by idiosyncratic structural factors (FIG. 22). Overall, these results indicate that both the binder and target interact with each other via the interfaces that w ere intended by design.

[0646] Example 20 - Binders are specific to their targets and are structurally diverse

[0647] Many practical applications require high target specificity, or absence of binding to unintended targets. To test the specificity of a subset of the identified SARS-CoV-2 RBD binders, their binding was measured against all the targets. All binders tested exhibit observable binding only to the intended target (FIG. 9).

[0648] Next, the structural diversity of the binder designs was analyzed to gain insight into how many independent solutions the method is able to generate for each design problem. Diversity is also practically important as it maximizes the chance that one of the designs will satisfy downstream requirements that are not know n in advance. The distribution of painvise TM-scores and secondary7structure content was observed across binding hits for the SARS- Atorney Docket No. 45288-0513WO1

[0649] CoV-2 RBD target. Compared to other active designs from RFdiffusion, the present designs were consistently lower in structural similarity to each other and had a higher frequency of allbeta structures.

[0650] Example 21 - Binders neutralize SARS-CoV-2 variant in live virus neutralization assays

[0651] Four of the binders (GDM SC2BRD 11, GDM SC2BRD 27, GDM SC2RBD 104, and GDM SC2BRD 50) were tested for the ability to neutralize SARS-CoV-2 infection. To measure neutralizing capabilities of binders, live-virus microneutralization assays were used on a Good Clinical Practice-compliant high-throughput platform, calibrated to WHO International Standards for anti-SARS-CoV-2 immunoglobulin against sequence-validated batches of four SARS-CoV-2 variants. It was found that all the binders were successful at neutralizing the ancestral (England2) strain with EC50 between 89-300 nM (FIGs. 18, 24). The 2-10 fold difference between the measured in vitro binding affinity (KD) and the cellular EC50 for virus neutralization is similar to what has been measured in the same assay for recombinant monoclonal antibodies used in clinical practice, such as Sotrovimab (KD = 210 nM, EC50 = 670 nM).

[0652] Interestingly, two of the binders (GDM SC2RBD 11 and GDM SC2RBD 129) were able to bind and neutralize three variants assayed, all four variants were neutralized by at least one designed binder, and the binder which showed the highest potency and lowest EC50 (GDM_SC2RBD_50) could only inhibit a single Ancestral / England2 variant.

[0653] Example 22 - Experimental structures of binder-target complexes confirm binding mode and structure

[0654] To validate the structures and binding modes of the designs, cryo-electron microscopy (crj'O-EM) was used to obtain structures of GDM SC2RBD 11, GDM SC2RBD 50, GDM SC2RBD 104, and GDM SC2RBD 129 in complex with the SARS-CoV-2 spike SI protein at 4.5 - 6.0 A resolution (FIGs. 19, 25A-25E). The experimental structures closely recapitulate the designed binder-target complexes, with binder Ca RMSDs of 0.84 - 3.14 A using the target S 1 protein as alignment reference.

[0655] D. Interleukin-7 receptor subunit alpha (IL7R-ot) protein

[0656] Methods Atorney Docket No. 45288-0513WO1

[0657] Purified protein stocks for IL7R-A (21-239) were purchased from BioTechne, with catalog number AVI10317. IL7R-A has C-terminal Fc and biotinylated Avi tags.

[0658] Yeast surface display and flow cytometry

[0659] Primary binding screen

[0660] Binder design sequences were codon-optimized by DNAworks and most were synthesized by Twist as gene fragments flanked by Bsal restriction sites as well as homology regions to a modified pETcon vector. Saccharomyces cerevisiae strain EBY100 cells (50 pL) were transformed using the lithium acetate method with 50 ng of linearized plasmid and a minimum of 10 ng of gene fragment insert in a 96-well plate. Cells were grown at 30 °C shaking at 1,000 rpm in complete synthetic medium -Trp -Ura + 2% glucose for 48-72 h. For protein expression, 2 x 107yeast cells were centrifuged at 1,800 x g for 5 mins at 20 °C and resuspended in 1 mL complete synthetic medium + 0.1% glucose + 2% galactose (SGCAA). Cells were incubated at 30 °C overnight (16-18 hours) and 8 x 106yeast cells were washed twice with 200 pL of lx PBS + 0.1% BSA (PBSF). centrifuged at 1,800 x g for 3 min at 20 °C and the supernatant was removed.

[0661] To screen for binding, yeast cells were then incubated with biotinylated target proteins (diluted in PBSF) for 1 hour, washed twice with PBSF and incubated with 25 ug / mL fluorescein isothiocyanate (FITC)-conjugated anti-Myc antibody (FITC-Ab) (Abeam) and 30 ug / mL streptavidin-phycoerythrin (SAPE. Thermo Fisher Scientific) for 30 minutes. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. Cells were analyzed on the CytoFlex LX (Beckman Coulter) or ZE5 Cell Analyzer (Bio-Rad) flow cytometers by measuring fluorescence of FITC and phycoerythrin (PE) to detect binder expression and target binding, respectively.

[0662] Interface mutation, competitive inhibition, and specificity experiments

[0663] Mutants were screened following the same method as the primary binding screen. For competition assays, yeast cells were incubated with biotinylated target proteins with or without a competitor protein for 1 h (the competitor protein was added to the biotinylated target protein master mix just before pipetting to the cells). The cells were then washed twice with PBSF and incubated with 25 pg / mL FITC-Ab (Abeam) and 30 pg / mL SAPE (Thermo Fisher Scientific) for 30 minutes. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. To test for specificity, specific designs as well as pooled cells were tested against target protein following the same method as the primary binding screen. Atorney Docket No. 45288-0513WO1

[0664] Designed binder expression and purification

[0665] Designed binders with the highest binding signal by yeast display were selected for E. coli expression and follow up experiments. Designs purchased as gene fragments were cloned into a modified pTriEx-4 vector containing an N-terminal 8-His tag using NEBridge Golden Gate cloning (NEB) at Bsal sites, transformed into DH5-a competent cells, miniprepped (Qiagen), and verified by Sanger sequencing (Azenta). A small number of designs were purchased from Twist Bioscience directly as cloned plasmids in pTriEx-4 or pET-29b. For expression, plasmids were transformed into BL21 (DE3) cells and the entire transformation mix inoculated into autoinduction medium consisting of Terrific Broth (TB) medium (Melford). 0.05% glucose, 0.2% alpha-lactose, and 50 p / mL carbenicillin. Cultures were incubated at 37 °C with shaking (220 or 1000 rpm) for 24 hours, harvested at 2,568 x g for 10 minutes, and pellets stored at -80 °C until purification. Cell pellets were lysed by chemical lysis using BugBuster Master Mix (Novagen) supplemented with complete EDTA-free protease inhibitor (Roche) with shaking for 20 minutes at room temperature. Lysates were clarified by centrifugation for 1 hour at 2.568 x g. then purified by immobilized metal affinity chromatography (IMAC) using Ni-NTA in either 0. 1 mL spin columns (Cytiva) or in HisPur™ Ni-NTA 96-well Spin Plates (Thermo Scientific), followed by size exclusion chromatography (SEC) on an AKTA Pure 25 M (Cytiva) equipped with an ALIAS autosampler (Spark Holland) using a Superdex 75 increase 10 / 300 GL column equilibrated in 20 mM sodium phosphate pH 7.5. Protein samples were analyzed by SDS-PAGE, and where required, concentrated using a 3 kDa MWCO Vivaspin concentrator (Cytiva). Protein concentrations were measured in triplicate by absorbance at 280 nm with a NanoDrop One (Thermo Scientific) using theoretical extinction coefficients, or using custom Python code. Purified proteins were aliquoted and stored at -80 pC until further use.

[0666] Where larger quantities of designed binders were required, for example for circular dichroism and x-ray crystallography experiments, expression was scaled up to 100-1000 mL BL21 (DE3) cultures and the above protocol followed with minor modifications. Cells were lysed using sonication for a total of 6 minutes (5 sec on / 5 sec off) on ice, and lysates were clarified by centrifugation at 40,000 x g for 45 minutes before being applied to 5 mL Ni-NTA column (Cytiva), followed by SEC.

[0667] Homogeneous Time Resolved Fluorescence (HTRF)

[0668] Measurement of binding affinity / binding dissociation constants (KD) Atorney Docket No. 45288-0513WO1

[0669] Binding affinities (KDS) were measured in equilibrium saturation-binding experiments with fixed binder design concentration and target titration. The total assay volume was 16 pL and all proteins and reagents were diluted in PPI europium detection buffer (Revvity). Target protein was premixed with HTRF acceptor reagent Streptavidin-d2 (Revvity), serially diluted, and transferred to a white ProxiPlate 384-shallow well microplate (‘assay plate’, Revvity ). Subsequently, 1 nM of each binder was added to the assay plate in duplicate (binders with KD < 0.5 nM were later re-assayed with 0.1 nM binder to ensure robust data fitting). The assay plate was centrifuged at 500 x g for 30 seconds, sealed and incubated at room temperature for between 30 minutes and 1 hour. HTRF donor mAb Anti-6HIS-Eu Gold (Revvity') was then added to a final concentration of 2 nM (lx), using a Mantis microfluidic liquid dispenser (Formulatrix) running software version 5.1.1 on Windows 10. The assay plate was centrifuged, sealed and incubated for a further 1 hour at room temperature.

[0670] HTRF signal was measured using a PHERAstar FSX (BMG) plate reader equipped with an HTRF 337 / 665 / 620 optic module running software version 5.70 R6 on Windows 10. The measurement conditions were as follows; 60 ps integration delay, 400 ps integration time, 60 flashes. The optimal focal (Z) height was determined using channel B for each experiment. HTRF ratios were calculated by dividing the acceptor signal at 665 nm by the donor signal at 620 nm and multiplying by a factor of 10,000. Mean background signal for each target-acceptor concentration (0 nM binder) was subtracted, and data were analyzed using custom Python code by fitting to the general 1 : 1 binding equation where R is the measured equilibrium HTRF signal, A and B are the titrated and fixed binding partner concentrations, respectively, and Rmax and KD are the fitted maximal HTRF signal and binding dissociation constants, respectively. This equation was used because some of the binders had KD values close to or lower than the fixed binder concentration used in the experiment, which causes the more common hyperbolic equation of 1 : 1 binding to overestimate the true KD. TO ensure reliable model fitting, a fixed binder concentration no more than 2-fold higher (and usually much lower) than the estimated KD was always used.

[0671] Bio-Layer Interferometry (BLI)

[0672] For selected controls and designs, KDS were measured by kinetic BLI assays to establish confidence in the HTRF results. Data w ere collected on the Octet R8 (Sartorius AG, Goettingen, Atorney Docket No. 45288-0513WO1

[0673] Germany) using the integrated Octet Discovery software version 12.2.2.20. Recombinant proteins were diluted from concentrated frozen stocks in 20 mM sodium phosphate pH 7.5, 0.05% Tween-20 (BLI buffer). A seven-point dilution series of the analyte protein was also prepared in BLI buffer to create a titration curve. Ni-NTA biosensors (Sartorius, catalog number 18-5102) were preequilibrated in BLI buffer for at least 10 minutes prior to starting the experiment. A fixed concentration of "ligand" (8His-tagged binder) was loaded onto sensors for 120-240 seconds, briefly washed for 10 seconds, followed by a 60 second baseline. Association of a titration series of "analyte" (target protein) was then performed for 90-420 seconds, followed by dissociation for 600-1200 seconds. All steps were performed at 25 °C and with shaking at 1000 rpm. Loading, association, and dissociation durations were optimized for each binder-target pair. Data were processed using Octet Analysis Studio (version 12.2.2.26). Measurements from reference sensors not loaded with ligand, as well as a reference well with 0 nM analyte, were subtracted from the final data to account for non-specific binding of analyte to the sensors and baseline drift due to unloading of ligand from sensors, respectively. Baseline (pre-association) signal was aligned to 0 before final analysis, where kinetic constants were obtained by nonlinear regression of 1: 1 or 2: 1 binding equations to the data. Fits were performed globally, over both association and dissociation, with a shared Rmax for all analyte concentrations.

[0674] Circular dichroism (CD) spectroscopy

[0675] Data were collected on a Jasco J-815 circular dichroism spectrometer, running software Spectra Manager Version 2.15.20, equipped with a PTC-348 temperature control device. Far- UV spectra (260-190 nm) and thermal unfolding measurements were recorded in 1 mm quartz glass cuvettes (Hellma) containing protein solutions at 10 pM in 20 mM sodium phosphate pH 7.5.

[0676] Baselines containing 20 mM sodium phosphate pH 7.5 were collected prior to sample analysis. Spectra were recorded in the far-UV (260-190 nm) at 20 °C with a scanning speed of 200 nm / min and a digital integration time (DIT) of 0.25 seconds. 25 accumulations (spectral scans) were recorded and automatically averaged by the software. Thermal unfolding data were recorded at 222 nm between 2-95 °C at a ramp rate of 2 °C / min, with measurements recorded at 0.2 °C intervals. The DIT was set to 4 seconds. Following thermal unfolding measurements, spectra in the far-UV were collected at 95 °C to measure CD spectra changes post thermal unfolding. Additional CD spectra were then collected following re-cooling of the same samples to 20 °C, to observe refolding. Atorney Docket No. 45288-0513WO1

[0677] Example 23 - Protein Design System

[0678] The proteins described herein were designed on a system comprising two components: 1) a "generator", or an all-atom diffusion-based generative model trained on protein structures and sequences from the Protein Data Bank (PDB) as well as a distillation set of AlphaFold predictions; and 2) a "filter" which scores generated designs to predict which are the most likely to succeed experimentally. To design binders, a structure of the "target" protein (e.g., IL7R-A) is input into the system and "hotspot" residues representing the target epitope are optionally designated, and the system then outputs a structure and sequence of a candidate binder for that target. A large number of designs are generated in silico and they are then filtered to a smaller set prior to experimental testing.

[0679] Example 24 - Picomolar-affinity binders from medium-throughput screening

[0680] Binders were designed against an IL7R-A target protein with diverse structural properties (FIG. 26, Table 7).

[0681] IL7R-A is a cell-surface receptor involved in lymphocyte development and a therapeutic target for acute lymphoblastic leukemia and HIV. The binding site of the native interleukin-7 ligand was targeted, which is moderately hydrophobic and subject to high success rates in previous design efforts.

[0682] [Table 7] - Binder design problem specifications for in silico benchmarking and experimental testing

[0683] Example 25 - Multiple binding hits within one 96-welI plate of designs per target

[0684] For the IL7R-A target, a large set of in silico designs 50-140 amino acids long was generated (Table 7) and a standardized filtering procedure was used to choose between 47 and 94 binder candidates to test for binding by yeast surface display. The present proteins were tested for the IL7R-A target protein, with the experimental success rate ranging from 9.6% to Atorney Docket No. 45288-0513WO1

[0685] Example 26 - State-of-the-art binding affinities on IL7Ra

[0686] High experimental success rates can reduce the labor and cost of obtaining binders, but once hits have been found, a far more important metric is binding affinity (KD) to the target. Most therapeutic antibodies have low-picomolar KDS, which is achieved by many rounds of experimental optimization. For binders used as research tools, low-nanomolar KD values or better is also typical. To measure how strongly the designed binders bound the IL7Ra target, yeast screening hits were recombinantly expressed and purified in E. coli to measure their KD values in vitro. Overall, 93% of designs chosen for follow up successfully expressed in E. coli (Table 8), and the majority were monodisperse by size-exclusion chromatography. A subset of designs assayed by circular dichroism (CD) spectroscopy all exhibited high thermostability (Tm > 95°C) and had the expected secondary' structures (FIGs. 27D, 28A-28B). For the recombinantly produced designs, KD values were measured using a homogeneous time- resolved fluorescence (HTRF) equilibrium saturation binding assay (FIGs. 27A-27C).

[0687] [Table 8] - Number of yeast hits successfully expressed in E. coli and tested for HTRF binding

[0688] Four total binders with picomolar KDS were identified for IL7Ra. Compared to the best unoptimized binders from other design methods, the proteins described herein had better KDS for the lL7Ra target protein by a margin of 10-fold. Taken together, the success rates and affinities of the present proteins suggest that it can generate binders for many potential applications using only one round of medium-throughput (such as one 96-well plate) screening and no further optimization (FIGs. 29A-29B).

[0689] Example 27 - Designs bind the intended epitope via the intended interactions

[0690] To test whether the designs bind the intended epitope on the target, binding was measured in the presence of a known competitive binder with the same target site. As expected, this reduced binding signal, with the reduction being smaller where the identified IL7Ra binders had a much higher affinity than the competitor. To test whether the IL7Ra binder designs bind their targets via the intended interactions, we measured binding of our top binders was measured after mutating 1-3 residues at the target-binding interface in their design models Atorney Docket No. 45288-0513WO1

[0691] (FIGs. 31A-31C). Almost all mutants had lower binding than their parent, suggesting successful disruption of the binding interface by the mutations. A small number of mutants had higher binding than the parent, but this can likely be explained by idiosyncratic structural factors (FIG. 30). Overall, these results indicate that both the binder and target interact with each other via the interfaces that were intended by design.

[0692] Example 28 - Binders are specific to their targets and are structurally diverse

[0693] Many practical applications require high target specificity, or absence of binding to unintended targets. To test the specificity of a subset of the identified IL7Ra binders, their binding was measured against all the targets. All binders tested exhibit observable binding only to the intended target (FIG. 9).

[0694] Next, the structural diversity of the binder designs was analyzed to gain insight into how many independent solutions the method is able to generate for each design problem. Diversity is also practically important as it maximizes the chance that one of the designs will satisfy dow nstream requirements that are not known in advance. The distribution of pairwise TM-scores and secondary7structure content was observed across binding hits for the IL7Ra target. Compared to the active designs from RF diffusion, the present designs were consistently lower in structural similarity to each other and had a higher frequency of all-beta structures.

[0695] E. Programmed death-ligand 1 (PD-L1) protein

[0696] Methods

[0697] Target protein expression and purification

[0698] Purified protein stocks for PD-Ll(19-239) were purchased from BioTechne. with catalog number AVI 156. PD-L1 has C-terminal Fc and biotinylated Avi tags.

[0699] Yeast surface display and flow cytometry Primary binding screen

[0700] Binder design sequences were codon-optimized by DNAworks and most were synthesized by Twist as gene fragments flanked by Bsal restriction sites as well as homology regions to a modified pETcon vector. Saccharomyces cerevisiae strain EBY100 cells (50 pL) were transformed using the lithium acetate method with 50 ng of linearized plasmid and a minimum of 1 ng of gene fragment insert in a 96-well plate. Cells were grown at 30 °C shaking Atorney Docket No. 45288-0513WO1 at 1,000 rpm in complete synthetic medium -Trp -Ura + 2% glucose for 48-72 h. For protein expression. 2 x 107yeast cells were centrifuged at 1,800 x g for 5 mins at 20 °C and resuspended in 1 mL complete synthetic medium + 0.1% glucose + 2% galactose (SGCAA). Cells were incubated at 30 °C overnight (16-18 hours) and 8 x 106yeast cells were washed twice with 200 pL of lx PBS + 0.1% BSA (PBSF), centrifuged at 1,800 x g for 3 min at 20 °C and the supernatant was removed.

[0701] To screen for binding, yeast cells were then incubated with biotinylated target proteins (diluted in PBSF) for 1 hour, washed twice with PBSF and incubated with 25 ug / mL fluorescein isothiocyanate (FITC)-conjugated anti-Myc antibody (FITC-Ab) (Abeam) and 30 ug / mL streptavidin-phycoerythrin (SAPE, Thermo Fisher Scientific) for 30 minutes. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. Cells were analyzed on the CytoFlex LX (Beckman Coulter) or ZE5 Cell Analyzer (Bio-Rad) flow cytometers by measuring fluorescence of FITC and phycoery thrin (PE) to detect binder expression and target binding, respectively.

[0702] Interface mutation, competitive inhibition, and specificity experiments

[0703] Mutants were screened following the same method as the primary binding screen. For competition assays, yeast cells were incubated with biotinylated target proteins with or without a competitor protein for 1 h (the competitor protein was added to the biotinylated target protein master mix just before pipetting to the cells). The cells were then washed twice with PBSF and incubated with 25 pg / mL FITC-Ab (Abeam) and 30 pg / mL SAPE (Thermo Fisher Scientific) for 30 minutes. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. To test for specificity, specific designs as well as pooled cells were tested against target protein following the same method as the primary’ binding screen.

[0704] Designed binder expression and purification

[0705] Designed binders with the highest binding signal by yeast display were selected for E. coli expression and follow up experiments. Designs purchased as gene fragments were cloned into a modified pTriEx-4 vector containing an N-terminal 8-His tag using NEBndge Golden Gate cloning (NEB) at Bsal sites, transformed into DH5-a competent cells, miniprepped (Qiagen), and verified by Sanger sequencing (Azenta). A small number of designs were purchased from Twist Bioscience directly as cloned plasmids in pTriEx-4 or pET-29b. For expression, plasmids were transformed into BL21 (DE3) cells and the entire transformation mix inoculated into autoinduction medium consisting of Terrific Broth (TB) medium (Melford), Atorney Docket No. 45288-0513WO1

[0706] 0.05% glucose, 0.2% alpha-lactose, and 50 p / mL carbenicillin. Cultures were incubated at 37 °C with shaking (220 or 1000 rpm) for 24 hours, harvested at 2,568 x g for 10 minutes, and pellets stored at -80 °C until purification. Cell pellets were lysed by chemical lysis using BugBuster Master Mix (Novagen) supplemented with complete EDTA-free protease inhibitor (Roche) with shaking for 20 minutes at room temperature. Lysates were clarified by centrifugation for 1 hour at 2,568 x g, then purified by immobilized metal affinity chromatography (IMAC) using Ni-NTA in either 0. 1 mL spin columns (Cytiva) or in HisPur™ Ni-NTA 96-well Spin Plates (Thermo Scientific), followed by size exclusion chromatography (SEC) on an AKTA Pure 25 M (Cytiva) equipped with an ALIAS autosampler (Spark Holland) using a Superdex 75 increase 10 / 300 GL column equilibrated in 20 mM sodium phosphate pH 7.5. Protein samples were analyzed by SDS-PAGE. and where required, concentrated using a 3 kDa MWCO Vivaspin concentrator (Cytiva). Protein concentrations were measured in triplicate by absorbance at 280 nm with a NanoDrop One (Thermo Scientific) using theoretical extinction coefficients, or using custom Python code. Purified proteins were aliquoted and stored at -80 pC until further use.

[0707] Where larger quantities of designed binders were required, for example for circular dichroism and x-ray crystallography experiments, expression was scaled up to 100-1000 mL BL21 (DE3) cultures and the above protocol followed with minor modifications. Cells were lysed using sonication for a total of 6 minutes (5 sec on / 5 sec off) on ice, and lysates were clarified by centrifugation at 40.000 x g for 45 minutes before being applied to 5 mL Ni-NTA column (Cytiva), followed by SEC.

[0708] Homogeneous Time Resolved Fluorescence (HTRF)

[0709] Measurement of binding affinity / binding dissociation constants (KD)

[0710] Binding affinities (KDS) were measured in equilibrium saturation-binding experiments with fixed binder design concentration and target titration. The total assay volume was 16 pL and all proteins and reagents w ere diluted in PPI europium detection buffer (Revvity). Target protein was premixed with HTRF acceptor reagent Streptavidin-d2 (Revvity), serially diluted, and transferred to a white ProxiPlate 384-shallow well microplate ("assay plate’. Revvity). Subsequently, 1 nM of each binder was added to the assay plate in duplicate (binders with KD < 0.5 nM were later re-assayed with 0.1 nM binder to ensure robust data fitting). The assay plate was centrifuged at 500 x g for 30 seconds, sealed and incubated at room temperature for between 30 minutes and 1 hour. HTRF donor mAb Anti-6HIS-Eu Gold (Revvity) was then added to a final concentration of 2 nM (lx), using a Mantis microfluidic liquid dispenser Atorney Docket No. 45288-0513WO1

[0711] (Formulatrix) running software version 5.1.1 on Windows 10. The assay plate was centrifuged, sealed and incubated for a further 1 hour at room temperature.

[0712] HTRF signal was measured using a PHERAstar FSX (BMG) plate reader equipped with an HTRF 337 / 665 / 620 optic module running software version 5.70 R6 on Windows 10. The measurement conditions w ere as follows; 60 ps integration delay, 400 ps integration time, 60 flashes. The optimal focal (Z) height was determined using channel B for each experiment. HTRF ratios were calculated by dividing the acceptor signal at 665 nm by the donor signal at 620 nm and multiplying by a factor of 10,000. Mean background signal for each target-acceptor concentration (0 nM binder) w as subtracted, and data were analyzed using custom Python code by fitting to the general 1 : 1 binding equation where R is the measured equilibrium HTRF signal, A and B are the titrated and fixed binding partner concentrations, respectively, and Rmax and KD are the fitted maximal HTRF signal and binding dissociation constants, respectively. This equation was used because some of the binders had KD values close to or lower than the fixed binder concentration used in the experiment, which causes the more common hyperbolic equation of 1 : 1 binding to overestimate the true KD. TO ensure reliable model fitting, a fixed binder concentration no more than 2-fold higher (and usually much lower) than the estimated KD was always used.

[0713] Bio-Laver Interferometry (BLI)

[0714] For selected controls and designs, KDS were measured by kinetic BLI assays to establish confidence in the HTRF results. Data were collected on the Octet R8 (Sartorius AG, Goettingen, Germany) using the integrated Octet Discovery' software version 12.2.2.20. Recombinant proteins were diluted from concentrated frozen stocks in 20 rnM sodium phosphate pH 7.5, 0.05% Tween-20 (BLI buffer). A seven-point dilution series of the analyte protein was also prepared in BLI buffer to create a titration curve. Ni-NTA biosensors (Sartorius, catalog number 18-5102) were preequilibrated in BLI buffer for at least 10 minutes prior to starting the experiment. A fixed concentration of "ligand" (8His-tagged binder) was loaded onto sensors for 120-240 seconds, briefly washed for 10 seconds, followed by a 60 second baseline. Association of a titration series of "analyte" (target protein) was then performed for 90-420 seconds, followed by dissociation for 600-1200 seconds. All steps were performed at 25 °C and w ith shaking at 1000 rpm. Loading, association, and dissociation durations were optimized Atorney Docket No. 45288-0513WO1 for each binder-target pair. Data were processed using Octet Analysis Studio (version 12.2.2.26). Measurements from reference sensors not loaded with ligand, as well as a reference well with 0 nM analyte, were subtracted from the final data to account for non-specific binding of analyte to the sensors and baseline drift due to unloading of ligand from sensors, respectively. Baseline (pre-association) signal was aligned to 0 before final analysis, where kinetic constants were obtained by nonlinear regression of 1: 1 or 2: 1 binding equations to the data. Fits were performed globally, over both association and dissociation, with a shared Rmax for all analyte concentrations.

[0715] Data were collected on a Jasco J-815 circular dichroism spectrometer, running software Spectra Manager Version 2.15.20, equipped with a PTC-348 temperature control device. Far- UV spectra (260-190 nm) and thermal unfolding measurements were recorded in 1 mm quartz glass cuvettes (Hellma) containing protein solutions at 10 pM in 20 mM sodium phosphate pH 7.5.

[0716] Baselines containing 20 mM sodium phosphate pH 7.5 were collected prior to sample analysis. Spectra were recorded in the far-UV (260-190 nm) at 20 °C with a scanning speed of 200 nm / min and a digital integration time (DIT) of 0.25 seconds. 25 accumulations (spectral scans) were recorded and automatically averaged by the software. Thermal unfolding data were recorded at 222 nm between 2-95 °C at a ramp rate of 2 °C / min, with measurements recorded at 0.2 °C intervals. The DIT was set to 4 seconds. Following thermal unfolding measurements, spectra in the far-UV were collected at 95 °C to measure CD spectra changes post thermal unfolding. Additional CD spectra were then collected following re-cooling of the same samples to 20 °C, to observe refolding.

[0717] Example 29 - Protein Design System

[0718] The proteins described herein were designed on a system comprising two components: 1) a "generator", or an all-atom diffusion-based generative model trained on protein structures and sequences from the Protein Data Bank (PDB) as well as a distillation set of AlphaFold predictions; and 2) a "filter" which scores generated designs to predict which are the most likely to succeed experimentally. To design binders, a structure of the "target" protein (e.g., PD-L1) is input into the system and "hotspot" residues representing the target epitope are optionally designated, and the system then outputs a structure and sequence of a candidate binder for that Atorney Docket No. 45288-0513WO1 target. A large number of designs are generated in silico and they are then filtered to a smaller set prior to experimental testing.

[0719] Example 30 - Picomolar-affinity binders from medium-throughput screening

[0720] Binders were designed against a PD-L1 target protein with diverse structural properties (FIG. 32, Table 9)

[0721] Programmed Death-Ligand 1 (PD-L1) is a cell-surface receptor that controls immune cell proliferation and an important therapeutic target for cancer. The target site is flat and difficult to bind by small molecules and smaller proteins.

[0722] [Table 9] - Binder design problem specifications for in silico benchmarking and experimental testing

[0723] Example 31 - Multiple binding hits within one 96-well plate of designs per target

[0724] For the PD-L1 target, a large set of in silico designs 50-140 amino acids long was generated (Table 9) and a standardized filtering procedure was used to choose between 47 and 94 binder candidates to test for binding by yeast surface display. The proteins described herein were tested for the PD-L1 target protein, with the experimental success rate ranging from 9.6% to 98%. For PD-L1, additional rounds of up to 31 designs were tested with improved in silico filters or using an updated system. These follow up rounds generally saw higher success rates, for a maximum per-round success rate of 29% on PD-L1.

[0725] Example 32 - State-of-the-art binding affinities on PD-L1

[0726] High experimental success rates can reduce the labor and cost of obtaining binders, but once hits have been found, a far more important metric is binding affinity (KD) to the target. Most therapeutic antibodies have low-picomolar KDS, which is achieved by many rounds of experimental optimization. For binders used as research tools, low-nanomolar KD values or better is also typical. To measure how strongly the designed binders bound the PD-L1 target, yeast screening hits were recombinantly expressed and purified in E. coli to measure their KD Atorney Docket No. 45288-0513WO1 values in vitro. Overall, 93% of designs chosen for follow up successfully expressed in E. coli (Table 10), and the majority were monodisperse by size-exclusion chromatography. A subset of designs assayed by circular dichroism (CD) spectroscopy all exhibited high thermostability (Tm > 95°C) and had the expected secondary structures (FIGs. 33D, 34A-34B). For the recombinantly produced designs, KD values were measured using a homogeneous time- resolved fluorescence (HTRF) equilibrium saturation binding assay (FIGs. 33A-33C).

[0727] [Table 10] - Number of yeast hits successfully expressed in E. coli and tested for HTRF binding

[0728] Two total binders with picomolar KDS were identified for PD-L1. Compared to the best unoptimized binders from other design methods, the KDS for the proteins described herein were better on the PD-L1 target protein, by a margin of 5 -fold. Taken together, the success rates and affinities achieved suggest that it can generate binders for many potential applications using only one round of medium-throughput (such as one 96-well plate) screening and no further optimization (FIGs. 35A-35B).

[0729] Example 33 - Designs bind the intended epitope via the intended interactions

[0730] To test whether the designs bind the intended epitope on the target, binding was measured in the presence of a known competitive binder with the same target site. As expected, this reduced binding signal, with the reduction being smaller where the identified PD-L1 binders had a much higher affinity than the competitor. To test whether the PD-L1 binder designs bind their targets via the intended interactions, we measured binding of our top binders was measured after mutating 1-3 residues at the target-binding interface in their design models (FIG. 37). Almost all mutants had lower binding than their parent, suggesting successful disruption of the binding interface by the mutations. A small number of mutants had higher binding than the parent, but this can likely be explained by idiosyncratic structural factors (FIG. 36). Overall, these results indicate that both the binder and target interact with each other via the interfaces that were intended by design. Atorney Docket No. 45288-0513WO1

[0731] Example 34 - Binders are specific to their targets and are structurally diverse

[0732] Many practical applications require high target specificity, or absence of binding to unintended targets. To test the specificity of a subset of the identified PD-L1 binders, their binding was measured against all the targets. All binders tested exhibit observable binding only to the intended target (FIG. 9).

[0733] Next, the structural diversity' of the binder designs was analyzed to gain insight into how many independent solutions the method is able to generate for each design problem. Diversity is also practically important as it maximizes the chance that one of the designs will satisfy downstream requirements that are not known in advance. The distribution of pairwise TM-scores and secondary structure content was observed across binding hits for the PD-L1 target. Compared to the active designs from RFdiffusion, the present proteins were consistently low er in structural similarity to each other and had a higher frequency of all-beta structures.

[0734] F. Tropomyosin receptor kinase A (Trk-A) protein

[0735] Methods

[0736] Target protein expression and purification

[0737] Purified protein stocks for Trk-A(34-423) were purchased from BioTechne, with catalog number AVI11378. Trk-A have C-terminal Fc and biotinylated Avi tags.

[0738] Yeast surface display and flow cytometry

[0739] Primary binding screen

[0740] Binder design sequences were codon-optimized by DNAworks and most were synthesized by Twist as gene fragments flanked by Bsal restriction sites as well as homology regions to a modified pETcon vector. Saccharomyces cerevisiae strain EBY100 cells (50 pL) were transformed using the lithium acetate method with 50 ng of linearized plasmid and a minimum of 10 ng of gene fragment insert in a 96-well plate. Cells were grown at 30 °C shaking at 1 ,000 rpm in complete synthetic medium -Trp -Ura + 2% glucose for 48-72 h. For protein expression, 2 x 107yeast cells were centrifuged at 1,800 x g for 5 mins at 20 °C and resuspended in 1 mL complete synthetic medium + 0.1% glucose + 2% galactose (SGCAA). Cells were incubated at 30 °C overnight (16-18 hours) and 8 x 106yeast cells were washed twice with 200 pL of lx PBS + 0.1% BSA (PBSF), centrifuged at 1,800 x g for 3 min at 20 °C and the supernatant was removed. Atorney Docket No. 45288-0513WO1

[0741] To screen for binding, yeast cells were then incubated with biotinylated target proteins (diluted in PBSF) for 1 hour, washed twice with PBSF and incubated with 25 ug / mL fluorescein isothiocyanate (FITC)-conjugated anti-Myc antibody (FITC-Ab) (Abeam) and 30 ug / mL streptavidin-phycoerythrin (SAPE, Thermo Fisher Scientific) for 30 minutes. Following binding, cells were w ashed once with PBSF and resuspended in 200 pL of PBSF. Cells were analyzed on the CytoFlex LX (Beckman Coulter) or ZE5 Cell Analyzer (Bio-Rad) flow cytometers by measuring fluorescence of FITC and phycoerythrin (PE) to detect binder expression and target binding, respectively.

[0742] Interface mutation, competitive inhibition, and specificity experiments

[0743] Mutants were screened following the same method as the primary binding screen. For competition assays, yeast cells were incubated with biotinylated target proteins with or without a competitor protein for 1 h (the competitor protein was added to the biotinylated target protein master mix just before pipetting to the cells). The cells w ere then washed twice with PBSF and incubated with 25 pg / mL FITC-Ab (Abeam) and 30 pg / mL SAPE (Thermo Fisher Scientific) for 30 minutes. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. To test for specificity, specific designs as well as pooled cells were tested against target protein following the same method as the primary binding screen.

[0744] Designed binder expression and purification

[0745] Designed binders with the highest binding signal by yeast display were selected for E. coli expression and follow^ up experiments. Designs purchased as gene fragments were cloned into a modified pTriEx-4 vector containing an N-terminal 8-His tag using NEBridge Golden Gate cloning (NEB) at Bsal sites, transformed into DH5-a competent cells, miniprepped (Qiagen), and verified by Sanger sequencing (Azenta). A small number of designs were purchased from Twist Bioscience directly as cloned plasmids in pTriEx-4 or pET-29b. For expression, plasmids were transformed into BL21 (DE3) cells and the entire transformation mix inoculated into autoinduction medium consisting of Terrific Broth (TB) medium (Melford). 0.05% glucose, 0.2% alpha-lactose, and 50 p / mL carbenicillin. Cultures were incubated at 37 °C with shaking (220 or 1000 rpm) for 24 hours, harvested at 2,568 x g for 10 minutes, and pellets stored at -80 °C until purification. Cell pellets were lysed by chemical lysis using BugBuster Master Mix (Novagen) supplemented with Complete EDTA-free protease inhibitor (Roche) with shaking for 20 minutes at room temperature. Lysates were clarified by centrifugation for 1 hour at 2,568 x g, then purified by immobilized metal affinity Atorney Docket No. 45288-0513WO1 chromatography (IMAC) using Ni-NTA in either 0. 1 mL spin columns (Cytiva) or in HisPur™ Ni-NTA 96-well Spin Plates (Thermo Scientific), followed by size exclusion chromatography (SEC) on an AKTA Pure 25 M (Cytiva) equipped with an ALIAS autosampler (Spark Holland) using a Superdex 75 increase 10 / 300 GL column equilibrated in 20 mM sodium phosphate pH 7.5. Protein samples were analyzed by SDS-PAGE, and where required, concentrated using a 3 kDa MWCO Vivaspin concentrator (Cytiva). Protein concentrations were measured in triplicate by absorbance at 280 nm with a NanoDrop One (Thermo Scientific) using theoretical extinction coefficients, or using custom Python code. Purified proteins were aliquoted and stored at -80 pC until further use.

[0746] Where larger quantities of designed binders were required, for example for circular dichroism and x-ray crystallography experiments, expression was scaled up to 100-1000 mL BL21 (DE3) cultures and the above protocol followed with minor modifications. Cells were lysed using sonication for a total of 6 minutes (5 sec on / 5 sec off) on ice, and lysates were clarified by centrifugation at 40,000 x g for 45 minutes before being applied to 5 mL Ni-NTA column (Cytiva). followed by SEC.

[0747] Homogeneous Time Resolved Fluorescence (HTRF)

[0748] Measurement of binding affinity binding dissociation constants (KD)

[0749] Binding affinities (KDS) were measured in equilibrium saturation-binding experiments with fixed binder design concentration and target titration. The total assay volume was 16 pL and all proteins and reagents were diluted in PPI europium detection buffer (Revvity). Target protein was premixed with HTRF acceptor reagent Streptavidin-d2 (Revvity), serially diluted, and transferred to a white ProxiPlate 384-shallow well microplate (‘assay plate’, Revvity). Subsequently, 1 nM of each binder was added to the assay plate in duplicate (binders with KD < 0.5 nM were later re-assayed with 0.1 nM binder to ensure robust data fitting). The assay plate was centrifuged at 500 x g for 30 seconds, sealed and incubated at room temperature for between 30 minutes and 1 hour. HTRF donor mAb Anti-6HIS-Eu Gold (Revvity) was then added to a final concentration of 2 nM (lx), using a Mantis microfluidic liquid dispenser (Formulatrix) running software version 5. 1. 1 on Windows 10. The assay plate was centrifuged, sealed and incubated for a further 1 hour at room temperature.

[0750] HTRF signal was measured using a PHERAstar FSX (BMG) plate reader equipped with an HTRF 337 / 665 / 620 optic module running software version 5.70 R6 on Windows 10. The measurement conditions were as follows; 60 ps integration delay, 400 ps integration time, 60 flashes. The optimal focal (Z) height was determined using channel B for each experiment. Atorney Docket No. 45288-0513WO1

[0751] HTRF ratios were calculated by dividing the acceptor signal at 665 nm by the donor signal at 620 nm and multiplying by a factor of 10,000. Mean background signal for each target-acceptor concentration (0 nM binder) was subtracted, and data were analyzed using custom Python code by fitting to the general 1 : 1 binding equation where R is the measured equilibrium HTRF signal, A and B are the titrated and fixed binding partner concentrations, respectively, and Rmax and KD are the fitted maximal HTRF signal and binding dissociation constants, respectively. This equation was used because some of the binders had KD values close to or lower than the fixed binder concentration used in the experiment, which causes the more common hyperbolic equation of 1 : 1 binding to overestimate the true KD. TO ensure reliable model fitting, a fixed binder concentration no more than 2-fold higher (and usually much lower) than the estimated KD was always used.

[0752] For selected controls and designs, KDS were measured by kinetic BLI assays to establish confidence in the HTRF results. Data were collected on the Octet R8 (Sartorius AG, Goettingen. Germany) using the integrated Octet Discovery software version 12.2.2.20. Recombinant proteins were diluted from concentrated frozen stocks in 20 rnM sodium phosphate pH 7.5, 0.05% Tween-20 (BLI buffer). A seven-point dilution series of the analyte protein was also prepared in BLI buffer to create a titration curve. Ni-NTA biosensors (Sartorius, catalog number 18-5102) were preequilibrated in BLI buffer for at least 10 minutes prior to starting the experiment. A fixed concentration of "ligand" (8His-tagged binder) was loaded onto sensors for 120-240 seconds, briefly washed for 10 seconds, followed by a 60 second baseline. Association of a titration series of "analyte" (target protein) was then performed for 90-420 seconds, followed by dissociation for 600-1200 seconds. All steps were performed at 25 °C and with shaking at 1000 rpm. Loading, association, and dissociation durations were optimized for each binder-target pair. Data were processed using Octet Analysis Studio (version 12.2.2.26). Measurements from reference sensors not loaded with ligand, as w ell as a reference well with 0 nM analyte, were subtracted from the final data to account for non-specific binding of analyte to the sensors and baseline drift due to unloading of ligand from sensors, respectively. Baseline (pre-association) signal w as aligned to 0 before final analysis, where kinetic constants were obtained by nonlinear regression of 1: 1 or 2: 1 binding equations to the data. Fits were Atorney Docket No. 45288-0513WO1 performed globally, over both association and dissociation, with a shared Rmax for all analyte concentrations.

[0753] Circular dichroism (CD) spectroscopy

[0754] Data were collected on a Jasco J-815 circular dichroism spectrometer, running software Spectra Manager Version 2.15.20, equipped with a PTC-348 temperature control device. Far- UV spectra (260-190 nm) and thermal unfolding measurements were recorded in 1 mm quartz glass cuvettes (Hellma) containing protein solutions at 10 pM in 20 mM sodium phosphate pH 7.5.

[0755] Baselines containing 20 mM sodium phosphate pH 7.5 were collected prior to sample analysis. Spectra were recorded in the far-UV (260-190 nm) at 20 °C with a scanning speed of 200 nm / min and a digital integration time (DIT) of 0.25 seconds. 25 accumulations (spectral scans) were recorded and automatically averaged by the software. Thermal unfolding data were recorded at 222 nm between 2-95 °C at a ramp rate of 2 °C / min, with measurements recorded at 0.2 °C intervals. The DIT was set to 4 seconds. Following thermal unfolding measurements, spectra in the far-UV were collected at 95 °C to measure CD spectra changes post thermal unfolding. Additional CD spectra were then collected following re-cooling of the same samples to 20 °C, to observe refolding.

[0756] Example 35 - Protein Design System

[0757] The proteins described herein were designed on a system comprising two components: 1) a "generator", or an all-atom diffusion-based generative model trained on protein structures and sequences from the Protein Data Bank (PDB) as well as a distillation set of AlphaFold predictions; and 2) a "filter" which scores generated designs to predict which are the most likely to succeed experimentally. To design binders, a structure of the "target" protein (e.g., Trk-A) is input into the system and "hotspot" residues representing the target epitope are optionally designated, and the system then outputs a structure and sequence of a candidate binder for that target. A large number of designs are generated in silico and they are then filtered to a smaller set prior to experimental testing.

[0758] Example 36 - Picomolar-affinity binders from medium-throughput screening

[0759] Binders were designed against a Trk-A target protein with diverse structural properties

[0760] (FIG. 38, Table 11) Atorney Docket No. 45288-0513WO1

[0761] Tropomyosin Receptor Kinase A (Trk-A) is a nen e growth factor receptor involved in autoimmune disease and an analgesic target for treating chronic pain. A hydrophobic pocket addressed by previous design efforts was targeted.

[0762] [Table 11] - Binder design problem specifications for in silico benchmarking and experimental testing

[0763] Example 37 - Multiple binding hits within one 96-well plate of designs per target

[0764] For the Trk-A target, a large set of in silico designs 50-140 amino acids long was generated (Table 11) and a standardized filtering procedure was used to choose between 47 and 94 binder candidates to test for binding by yeast surface display. Proteins targeting Trk-A were evaluated, with the experimental success rate ranging from 9.6% to 98%.

[0765] Example 38 - State-of-the-art binding affinities on Trk-A

[0766] High experimental success rates can reduce the labor and cost of obtaining binders, but once hits have been found, a far more important metric is binding affinity (KD) to the target. Most therapeutic antibodies have low-picomolar KDS, which is achieved by many rounds of experimental optimization. For binders used as research tools, low-nanomolar KD values or better is also typical. To measure how strongly the designed binders bound the Trk-A target, yeast screening hits were recombinantly expressed and purified in E. coli to measure their KD values in vitro. Overall, 93% of designs chosen for follow up successfully expressed in E. coli (Table 12), and the majority were monodisperse by size-exclusion chromatography. A subset of designs assayed by circular dichroism (CD) spectroscopy all exhibited high thermostability (Tm > 95°C) and had the expected secondary structures (FIGs. 39D, 40A-40B). For the recombinantly produced designs, KD values were measured using a homogeneous time- resolved fluorescence (HTRF) equilibrium saturation binding assay (FIGs. 39A-39C). Atorney Docket No. 45288-0513WO1

[0767] [Table 12] - Number of yeast hits successfully expressed in E. coli and tested for HTRF binding

[0768] Compared to the best unoptimized binders from other design methods, KDS for the proteins described herein were better on the Trk-A target protein, by a margin of 100-fold. Taken together, the success rates and affinities achieved suggest that this method can generate binders for many potential applications using only one round of medium-throughput (such as one 96-well plate) screening and no further optimization (FIG. 41).

[0769] Example 39 - Designs bind the intended epitope via the intended interactions

[0770] To test whether the designs bind the intended epitope on the target, binding was measured in the presence of a known competitive binder with the same target site. As expected, this reduced binding signal, with the reduction being smaller where the identified Trk-A binders had a much higher affinity than the competitor. To test whether the Trk-A binder designs bind their targets via the intended interactions, we measured binding of our top binders was measured after mutating 1-3 residues at the target-binding interface in their design models (FIGs. 43A-43B). Almost all mutants had lower binding than their parent, suggesting successful disruption of the binding interface by the mutations. A small number of mutants had higher binding than the parent, but this can likely be explained by idiosyncratic structural factors (FIG. 42). Overall, these results indicate that both the binder and target interact with each other via the interfaces that were intended by design.

[0771] Example 40 - Binders are specific to their targets and are structurally diverse

[0772] Many practical applications require high target specificity, or absence of binding to unintended targets. To test the specificity of a subset of the identified Trk-A binders, their binding was measured against all the targets. All binders tested exhibit observable binding only to the intended target (FIG. 9).

[0773] Next, the structural diversity of the binder designs was analyzed to gain insight into how many independent solutions the method is able to generate for each design problem. Diversity is also practically important as it maximizes the chance that one of the designs will Atorney Docket No. 45288-0513WO1 satisfy downstream requirements that are not known in advance. The distribution of pairwise TM-scores and secondary structure content was observed across binding hits for the Trk-A target. Compared to the active designs from RFdiffusion, the present proteins were consistently lower in structural similarity to each other and had a higher frequency of all-beta structures.

[0774] G. Interleukin- 17 (IL-17A) protein

[0775] Methods

[0776] Target protein expression and purification

[0777] Purified protein stocks for IL-17A(24-155) were purchased from BioTechne, with catalog number BT7955. IL-17A is biotinylated via sugars, and IL-17A is a disulfide-linked homo-dimer.

[0778] Yeast surface display and flow cytometry

[0779] Primary binding screen

[0780] Binder design sequences were codon-optimized by DNAworks and most were synthesized by Twist as gene fragments flanked by Bsal restriction sites as well as homology regions to a modified pETcon vector. Saccharomyces cerevisiae strain EBY100 cells (50 pL) were transformed using the lithium acetate method with 50 ng of linearized plasmid and a minimum of 10 ng of gene fragment insert in a 96-well plate. Cells were grown at 30 °C shaking at 1,000 rpm in complete synthetic medium -Trp -Ura + 2% glucose for 48-72 h. For protein expression, 2 x 107yeast cells were centrifuged at 1,800 x g for 5 mins at 20 °C and resuspended in 1 mL complete synthetic medium + 0.1% glucose + 2% galactose (SGCAA). Cells were incubated at 30 °C overnight (16-18 hours) and 8 x 106yeast cells were washed twice with 200 pL of lx PBS + 0.1% BSA (PBSF). centrifuged at 1,800 x g for 3 min at 20 °C and the supernatant was removed.

[0781] To screen for binding, yeast cells were then incubated with biotinylated target proteins (diluted in PBSF) for 1 hour, washed twice with PBSF and incubated with 25 ug / mL fluorescein isothiocyanate (FITC)-conjugated anti-Myc antibody (FITC-Ab) (Abeam) and 30 ug / mL streptavidin-phycoerythrin (SAPE, Thermo Fisher Scientific) for 30 minutes. For IL- 17A, the signal was increased by binding with avidity; target proteins were pre-incubated with 25 ug / mL FITC-Ab and 30 ug / mL SAPE for 30 minutes before incubating with cells. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. Cells were analyzed on the CytoFlex LX (Beckman Coulter) or ZE5 Cell Analyzer (Bio-Rad) Atorney Docket No. 45288-0513WO1 flow cytometers by measuring fluorescence of FITC and phycoerythrin (PE) to detect binder expression and target binding, respectively.

[0782] Interface mutation, competitive inhibition, and specificity’ experiments

[0783] Mutants were screened following the same method as the primary' binding screen. For competition assays, yeast cells were incubated with biotinylated target proteins with or without a competitor protein for 1 h (the competitor protein was added to the biotinylated target protein master mix just before pipetting to the cells). The cells were then washed twice with PBSF and incubated with 25 pg / mL FITC-Ab (Abeam) and 30 pg / mL SAPE (Thermo Fisher Scientific) for 30 minutes. For IL-17A, the signal was increased by binding with avidity' similar to the primary binding screen; target proteins were pre-incubated with 25 ug / mL FITC-Ab and 30 ug / mL SAPE for 30 minutes before adding competitor protein and incubating with cells. Following binding, cells were washed once with PBSF and resuspended in 200 pL of PBSF. To test for specificity , specific designs as well as pooled cells were tested against target protein following the same method as the primary binding screen.

[0784] Designed binder expression and purification

[0785] Designed binders with the highest binding signal by yeast display were selected for E. coli expression and follow up experiments. Designs purchased as gene fragments were cloned into a modified pTriEx-4 vector containing an N-terminal 8-His tag using NEBridge Golden Gate cloning (NEB) at Bsal sites, transformed into DH5-a competent cells, miniprepped (Qiagen), and verified by Sanger sequencing (Azenta). A small number of designs were purchased from Twist Bioscience directly as cloned plasmids in pTriEx-4 or pET-29b. For expression, plasmids were transformed into BL21 (DE3) cells and the entire transformation mix inoculated into autoinduction medium consisting of Terrific Broth (TB) medium (Melford), 0.05% glucose, 0.2% alpha-lactose, and 50 p / mL carbenicillin. Cultures were incubated at 37 °C with shaking (220 or 1000 rpm) for 24 hours, harvested at 2,568 x g for 10 minutes, and pellets stored at -80 °C until purification. Cell pellets were lysed by chemical lysis using BugBuster Master Mix (Novagen) supplemented with complete EDTA-free protease inhibitor (Roche) with shaking for 20 minutes at room temperature. Lysates were clarified by centrifugation for 1 hour at 2,568 x g, then purified by immobilized metal affinity chromatography (IMAC) using Ni-NTA in either 0. 1 mL spin columns (Cytiva) or in HisPur™ Ni-NTA 96-well Spin Plates (Thermo Scientific), followed by size exclusion chromatography (SEC) on an AKTA Pure 25 M (Cytiva) equipped with an ALIAS autosampler (Spark Holland) Atorney Docket No. 45288-0513WO1 using a Superdex 75 increase 10 / 300 GL column equilibrated in 20 mM sodium phosphate pH 7.5. Protein samples were analyzed by SDS-PAGE. and where required, concentrated using a 3 kDa MWCO Vivaspin concentrator (Cytiva). Protein concentrations were measured in triplicate by absorbance at 280 nm with a NanoDrop One (Thermo Scientific) using theoretical extinction coefficients, or using custom Python code. Purified proteins were aliquoted and stored at -80 pC until further use.

[0786] Where larger quantities of designed binders were required, for example for circular dichroism and x-ray crystallography experiments, expression was scaled up to 100-1000 mL BL21 (DE3) cultures and the above protocol followed with minor modifications. Cells were lysed using sonication for a total of 6 minutes (5 sec on / 5 sec off) on ice, and lysates were clarified by centrifugation at 40.000 x g for 45 minutes before being applied to 5 mL Ni-NTA column (Cytiva), followed by SEC.

[0787] Homogeneous Time Resolved Fluorescence (HTRF)

[0788] Measurement of binding affinity / binding dissociation constants (KD)

[0789] Binding affinities (KDS) were measured in equilibrium saturation-binding experiments with fixed binder design concentration and target titration. The total assay volume was 16 pL and all proteins and reagents were diluted in PPI europium detection buffer (Revvity). Target protein was premixed with HTRF acceptor reagent Streptavidin-d2 (Revvity), serially diluted, and transferred to a white ProxiPlate 384-shallow well microplate (‘assay plate', Revvity). Subsequently, 1 nM of each binder was added to the assay plate in duplicate (binders with KD < 0.5 nM were later re-assayed with 0.1 nM binder to ensure robust data fitting). The assay plate was centrifuged at 500 x g for 30 seconds, sealed and incubated at room temperature for between 30 minutes and 1 hour. HTRF donor mAb Anti-6HIS-Eu Gold (Revvity ) was then added to a final concentration of 2 nM (lx), using a Mantis microfluidic liquid dispenser (Formulatrix) running software version 5.1.1 on Windows 10. The assay plate was centrifuged, sealed and incubated for a further 1 hour at room temperature.

[0790] HTRF signal was measured using a PHERAstar FSX (BMG) plate reader equipped wi th an HTRF 337 / 665 / 620 optic module running software version 5.70 R6 on Windows 10. The measurement conditions were as follows; 60 ps integration delay, 400 ps integration time, 60 flashes. The optimal focal (Z) height was determined using channel B for each experiment. HTRF ratios were calculated by dividing the acceptor signal at 665 nm by the donor signal at 620 nm and multiplying by a factor of 10,000. Mean background signal for each target-acceptor Atorney Docket No. 45288-0513WO1 concentration (0 nM binder) was subtracted, and data were analyzed using custom Python code by fitting to the general 1 : 1 binding equation where R is the measured equilibrium HTRF signal, A and B are the titrated and fixed binding partner concentrations, respectively, and Rmax and KD are the fitted maximal HTRF signal and binding dissociation constants, respectively. This equation was used because some of the binders had KD values close to or lower than the fixed binder concentration used in the experiment, which causes the more common hyperbolic equation of 1 : 1 binding to overestimate the true KD. TO ensure reliable model fitting, a fixed binder concentration no more than 2-fold higher (and usually much lower) than the estimated KD was always used.

[0791] Bio-Laver Interferometry (BL1)

[0792] For selected controls and designs, KDS were measured by kinetic BLI assays to establish confidence in the HTRF results. Data were collected on the Octet R8 (Sartorius AG, Goettingen, Germany) using the integrated Octet Discovery software version 12.2.2.20. Recombinant proteins were diluted from concentrated frozen stocks in 20 mM sodium phosphate pH 7.5, 0.05% Tween-20 (BLI buffer). A seven-point dilution series of the analyte protein was also prepared in BLI buffer to create a titration curve. Ni-NTA biosensors (Sartorius, catalog number 18-5102) were preequilibrated in BLI buffer for at least 10 minutes prior to starting the experiment. A fixed concentration of "ligand" (8His-tagged binder) was loaded onto sensors for 120-240 seconds, briefly washed for 10 seconds, followed by a 60 second baseline. Association of a titration series of "analyte" (target protein) was then performed for 90-420 seconds, followed by dissociation for 600-1200 seconds. All steps were performed at 25 °C and with shaking at 1000 rpm. Loading, association, and dissociation durations were optimized for each binder-target pair. Data were processed using Octet Analysis Studio (version 12.2.2.26). Measurements from reference sensors not loaded with ligand, as well as a reference well with 0 nM analyte, w ere subtracted from the final data to account for non-specific binding of analyte to the sensors and baseline drift due to unloading of ligand from sensors, respectively. Baseline (pre-association) signal was aligned to 0 before final analysis, where kinetic constants were obtained by nonlinear regression of 1 : 1 or 2: 1 binding equations to the data. Fits were performed globally, over both association and dissociation, with a shared Rmax for all analyte concentrations. Atorney Docket No. 45288-0513WO1

[0793] Data were collected on a Jasco J-815 circular dichroism spectrometer, running software Spectra Manager Version 2.15.20, equipped with a PTC-348 temperature control device. Far- UV spectra (260-190 nm) and thermal unfolding measurements were recorded in 1 mm quartz glass cuvettes (Hellma) containing protein solutions at 10 pM in 20 mM sodium phosphate pH 7.5.

[0794] Baselines containing 20 mM sodium phosphate pH 7.5 were collected prior to sample analysis. Spectra were recorded in the far-UV (260-190 nm) at 20 °C with a scanning speed of 200 nm / min and a digital integration time (DIT) of 0.25 seconds. 25 accumulations (spectral scans) were recorded and automatically averaged by the software. Thermal unfolding data were recorded at 222 nm between 2-95 °C at a ramp rate of 2 °C / min, with measurements recorded at 0.2 °C intervals. The DIT was set to 4 seconds. Following thermal unfolding measurements, spectra in the far-UV were collected at 95 °C to measure CD spectra changes post thermal unfolding. Additional CD spectra were then collected following re-cooling of the same samples to 20 °C. to observe refolding.

[0795] Example 41 - Protein Design System

[0796] The proteins described herein were designed on a system comprising two components: 1) a "generator", or an all-atom diffusion-based generative model trained on protein structures and sequences from the Protein Data Bank (PDB) as well as a distillation set of AlphaFold predictions; and 2) a "filter" which scores generated designs to predict which are the most likely to succeed experimentally. To design binders, a structure of the "target" protein (e.g., IL-17A) is input into the system and "hotspot" residues representing the target epitope are optionally designated, and the system then outputs a structure and sequence of a candidate binder for that target. A large number of designs are generated in silico and they are then filtered to a smaller set prior to experimental testing.

[0797] Example 42 - Picomolar-affinity binders from medium-throughput screening

[0798] Binders were designed against an IL-17A target protein with diverse structural properties (FIG. 44, Table 13).

[0799] Interleu...

Claims

1. Atorney Docket No. 45288-0513WO1WHAT IS CLAIMED IS:

1. A protein comprising a Vascular Endothelial Growth Factor A (VEGF-A) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:

4. or SEQ ID NO: 5.

2. The protein of claim 1, w herein the protein comprises a sequence that is at least 85% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

3. The protein of claim 1, wherein the protein comprises a sequence that is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

4. The protein of claim 1, wherein the protein comprises a sequence that is at least 95% identical to SEQ ID NO:

2. SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

5. The protein of claim 1, wherein the protein comprises a sequence that is at least 99% identical to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

6. The protein of claim 1, wherein the protein comprises SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

7. The protein of claim 1. wherein the protein is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

8. The protein of any one of claims 1-7, wherein the protein is a single-chain protein.

9. The protein of any one of claims 1-8, wherein the protein binds to the extracellular domain of VEGF-A.

10. The protein of any one of claims 1-9, wherein protein binds to VEGF-A with a KD value of about 1 x 10‘9M.Atorney Docket No. 45288-0513WO111. The protein of any one of claims 1-10, wherein the protein binding to VEGF-A inhibits VEGF-A activity’.

12. A nucleic acid comprising a sequence encoding the protein of any one of claims 1-11.

13. A vector comprising the nucleic acid of claim 12.

14. A cell comprising the nucleic acid of claim 12, or the vector of claim 13.

15. A pharmaceutical composition comprising any one of the proteins of claims 1- 11, the nucleic acids of claim 12, the vector of claim 13, or the cell of claim 14; and a pharmaceutically acceptable carrier.

16. The protein of any one of claims 1-11, the nucleic acids of claim 12, the vector of claim 13, or the cell of claim 14, for use in therapy.

17. The protein of any one of claims 1-11. the nucleic acids of claim 12, the vector of claim 13, or the cell of claim 14, for use in treatment of a VEGF-A associated disease, preferably wherein the VEGF-A associated disease is a cancer, an inflammatory disease, a cardiovascular disease, or an autoimmune disease.

18. A method of treating a VEGF-A associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the protein of any one of claims 1-11, the nucleic acid of claim 12, the vector of claim 13, the cell of claim 14 or the pharmaceutical composition of claim 15.

19. The method of claim 18, wherein the subject is a human.

20. The method of claim 18 or 19, wherein the VEGF-A associated disease is a cancer, an inflammatory disease, a cardiovascular disease, or an autoimmune disease.Atorney Docket No. 45288-0513WO121. The method of claim 20, wherein the cancer is colorectal cancer, lung cancer, glioblastoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, or renal cell cancer.

22. The method of claim 20, wherein the cardiovascular disease is ischemic heart disease, heart failure, myocardial infarction, or coronary’ artery disease.

23. The method of claim 20, wherein the autoimmune disease is systemic lupus ery thematosus, rheumatoid arthritis, or multiple sclerosis.

24. A protein comprising an Epstein-Barr Virus BCL-2 homolog (BHRF1) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

25. The protein of claim 24. wherein the protein comprises a sequence that is at least 85% identical to SEQ ID NO:

7. SEQ ID NO:

8. or SEQ ID NO: 9.

26. The protein of claim 24, wherein the protein comprises a sequence that is at least 90% identical to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

27. The protein of claim 24, wherein the protein comprises a sequence that is at least 95% identical to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

28. The protein of claim 24. wherein the protein comprises a sequence that is at least 99% identical to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

29. The protein of claim 24, wherein the protein comprises SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

30. The protein of claim 24, wherein the protein is SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

31. The protein of any one of claims 24-30, wherein the protein is a single-chain protein.Atorney Docket No. 45288-0513WO132. The protein of any one of claims 24-31, wherein protein binds to BHRF1 with a KD value of about 1 x 10'9M.

33. The protein of any one of claims 24-32, wherein the protein binding to BHRF1 inhibits BHRF1 activity.

34. A nucleic acid comprising a sequence encoding the protein of any one of claims 24-33.

35. A vector comprising the nucleic acid of claim 34.

36. A cell comprising the nucleic acid of claim 34, or the vector of claim 35.

37. A pharmaceutical composition comprising any one of the proteins of claims 24- 33. the nucleic acids of claim 34, the vector of claim 35. or the cell of claim 36; and a pharmaceutically acceptable carrier.

38. The protein of any one of claims 24-33, the nucleic acid of claim 34, the vector of claim 35, or the cell of claim 36, for use in therapy.

39. The protein of any one of claims 24-33, the nucleic acid of claim 34, the vector of claim 35, or the cell of claim 36, for use in treatment of a BHRF1 associated disease, preferably wherein the BHRF1 associated disease is a cancer.

40. A method of treating a BHRF1 associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the protein of any one of claims 24-33, the nucleic acid of claim 34, the vector of claim 35, the cell of claim 36 or the pharmaceutical composition of claim 37.

41. The method of claim 40, wherein the subject is a human.

42. The method of claim 40 or 41 , wherein the BHRF 1 associated disease is a cancer.Atorney Docket No. 45288-0513WO143. The method of claim 42, wherein the cancer is colorectal cancer, lung cancer, glioblastoma, cervical cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, or renal cell cancer.

44. A protein comprising a Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) RBD protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

45. The protein of claim 44, wherein the protein comprises a sequence that is at least 85% identical to SEQ ID NO:

11. SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

46. The protein of claim 44, wherein the protein comprises a sequence that is at least 90% identical to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:

15. or SEQ ID NO: 16.

47. The protein of claim 44, wherein the protein comprises a sequence that is at least 95% identical to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:

15. or SEQ ID NO: 16.

48. The protein of claim 44, wherein the protein comprises a sequence that is at least 99% identical to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

49. The protein of claim 44, wherein the protein comprises SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

50. The protein of claim 44, wherein the protein is SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

51. The protein of any one of claims 44-50, wherein the protein is a single-chain protein.Atorney Docket No. 45288-0513WO152. The protein of any one of claims 44-51, wherein the protein binds to the extracellular domain of SARS-CoV-2 RBD.

53. The protein of any one of claims 44-52, wherein protein binds to SARS-CoV-2 RBD with a KD value of about 1 x 10'9M.

54. The protein of any one of claims 44-53, wherein the protein binding to SARS- CoV-2 RBD inhibits SARS-CoV-2 activity.

55. A nucleic acid comprising a sequence encoding the protein of any one of claims 44-54.

56. A vector comprising the nucleic acid of claim 55.

57. A cell comprising the nucleic acid of claim 55, or the vector of claim 56.

58. A pharmaceutical composition comprising any one of the proteins of claims 44- 54. the nucleic acids of claim 55, the vector of claim 56. or the cell of claim 57; and a pharmaceutically acceptable carrier.

59. The protein of any one of claims 44-54, the nucleic acid of claim 55, the vector of claim 56, or the cell of claim 57, for use in therapy.

60. The protein of any one of claims 44-54, the nucleic acid of claim 55, the vector of claim 57, or the cell of claim 58, for use in treatment of a SARS-CoV-2 associated disease, preferably wherein the SARS-CoV-2 associated disease is COVID 19.

61. A method of treating a SARS-CoV-2 associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the protein of any one of claims 44-54 or the pharmaceutical composition of claim 58.

62. The method of claim 61, wherein the subject is a human.Atorney Docket No. 45288-0513WO163. The method of claim 61 or 62, wherein the SARS-CoV-2 associated disease is COVID19.

64. A protein comprising an Interleukin-7 receptor subunit alpha (IL7R-a) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO:

18. SEQ ID NO:

19. or SEQ ID NO: 20.

65. The protein of claim 64, wherein the protein comprises a sequence that is at least 85% identical to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

66. The protein of claim 64, wherein the protein comprises a sequence that is at least 90% identical to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

67. The protein of claim 64. wherein the protein comprises a sequence that is at least 95% identical to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

68. The protein of claim 64, wherein the protein comprises a sequence that is at least 99% identical to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

69. The protein of claim 64, wherein the protein comprises SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

70. The protein of claim 64, wherein the protein is SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

71. The protein of any one of claims 64-70, wherein the protein is a single-chain protein.

72. The protein of any one of claims 64-71, wherein protein binds to IL7R-a with a KD value of about 1 x 1 O'9M.

73. The protein of any one of claims 64-72, wherein the protein binding to IL7R-a inhibits IL7R-a activity.Atorney Docket No. 45288-0513WO174. A nucleic acid comprising a sequence encoding the protein of any one of claims 64-73.

75. A vector comprising the nucleic acid of claim 74.

76. A cell comprising the nucleic acid of claim 74, or the vector of claim 75.

77. A pharmaceutical composition comprising any one of the proteins of claims 64- 73, the nucleic acids of claim 74, the vector of claim 75, or the cell of claim 76; and a pharmaceutically acceptable carrier.

78. The protein of any one of claims 64-73, the nucleic acid of claim 74, the vector of claim 75, or the cell of claim 76, for use in therapy.

79. The protein of any one of claims 64-73, the nucleic acid of claim 74, the vector of claim 75. or the cell of claim 76. for use in treatment of an IL7R-a associated disease, preferably wherein the IL7R-a associated disease is a cancer.

80. A method of treating an IL7R-a associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the protein of any one of claims 64-73 or the pharmaceutical composition of claim 77.

81. The method of claim 80, wherein the subj ect is a human.

82. The method of claim 80 or 81, wherein the IL7R-a associated disease is a cancer.

83. The method of claim 82, wherein the cancer is a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary7gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, prostate cancer, or combinations thereof.Atorney Docket No. 45288-0513WO184. A protein comprising a Programmed death-ligand 1 (PD-L1) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO:22, SEQ ID NO: 23, or SEQ ID NO: 24.

85. The protein of claim 84, wherein the protein comprises a sequence that is at least 85% identical to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

86. The protein of claim 84, wherein the protein comprises a sequence that is at least 90% identical to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

87. The protein of claim 84. wherein the protein comprises a sequence that is at least 95% identical to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

88. The protein of claim 84, wherein the protein comprises a sequence that is at least 99% identical to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

89. The protein of claim 84, wherein the protein comprises SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

90. The protein of claim 84, wherein the protein is SEQ ID NO: 22, SEQ ID NO:23, or SEQ ID NO: 24.

91. The protein of any one of claims 84-90, wherein the protein is a single-chain protein.

92. The protein of any one of claims 84-91, wherein protein binds to PD-L1 with a KD value of about 1 x 1 O'9M.

93. The protein of any one of claims 84-92, wherein the protein binding to PD-L1 inhibits PD-L1 activity.

94. A nucleic acid comprising a sequence encoding the protein of any one of claimsAtorney Docket No. 45288-0513WO195. A vector comprising the nucleic acid of claim 94.

96. A cell comprising the nucleic acid of claim 94, or the vector of claim 95.

97. A pharmaceutical composition comprising any one of the proteins of claims 84- 93, the nucleic acids of claim 94, the vector of claim 95, or the cell of claim 96; and a pharmaceutically acceptable carrier.

98. The protein of any one of claims 84-93, the nucleic acid of claim 94, the vector of claim 95, or the cell of claim 96, for use in therapy.

99. The protein of any one of claims 84-93, the nucleic acid of claim 94, the vector of claim 95, or the cell of claim 96, for use in treatment of a PD-L1 associated disease, preferably wherein the PD-L1 associated disease is a cancer, an inflammatory disease, an infectious disease, or an autoimmune disease.

100. A method of treating a PD-L1 associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the protein of any one of claims 84-93 or the pharmaceutical composition of claim 97.

101. The method of claim 100, wherein the subject is a human.

102. The method of claim 100 or 101, wherein the PD-L1 associated disease is a cancer, an inflammatory disease, an infectious disease, or an autoimmune disease.

103. The method of claim 102, wherein the cancer is a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary' gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, prostate cancer, or combinations thereof.Atorney Docket No. 45288-0513WO1104. The method of claim 102, wherein the inflammatory disease is acute inflammation, chronic inflammation, osteoarthritis, juvenile chronic arthritis, systemic vasculitis, systemic sclerosis, idiopathic inflammatory myopathies, sarcoidosis, idiopathic demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, non- viral hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, gluten-sensitive enteropathy, erythema multiforme, contact dermatitis, eosinophilic pneumonia, idiopathic pulmonary fibrosis, and graft-versus-host-disease.

105. The method of claim 102, wherein the infectious disease is a viral infection, a bacterial infection, or a fungal infection.

106. The method of claim 102, wherein the autoimmune disease is systemic lupus erythematosus, psoriasis, rheumatoid arthritis, or multiple sclerosis.

107. A protein comprising a Tropomyosin-receptor kinase A (Trk-A) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

108. The protein of claim 107, wherein the protein comprises a sequence that is at least 85% identical to SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

109. The protein of claim 107, wherein the protein comprises a sequence that is at least 90% identical to SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

110. The protein of claim 107, wherein the protein comprises a sequence that is at least 95% identical to SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

111. The protein of claim 107, wherein the protein comprises a sequence that is at least 99% identical to SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

112. The protein of claim 107, wherein the protein comprises SEQ ID NO: 26, SEQ ID NO:

27. or SEQ ID NO: 28.Atorney Docket No. 45288-0513WO1113. The protein of claim 107, wherein the protein is SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

114. The protein of any one of claims 107-113, wherein the protein is a single-chain protein.

115. The protein of any one of claims 107-114, wherein protein binds to Trk-A with a KD value of about 1 x 10'9M.

116. The protein of any one of claims 107-115, wherein the protein binding to Trk- A inhibits Trk-A activity.

117. A nucleic acid comprising a sequence encoding the protein of any one of claims 107-116.

118. A vector comprising the nucleic acid of claim 117.

119. A cell comprising the nucleic acid of claim 117, or the vector of claim 118.

120. A pharmaceutical composition comprising any one of the proteins of claims 107-11 , the nucleic acids of claim 1 17, the vector of claim 1 18, or the cell of claim 1 19; and a pharmaceutically acceptable carrier.

121. The protein of any one of claims 107-116, the nucleic acid of claim 117, the vector of claim 1 18, or the cell of claim 119, for use in therapy.

122. The protein of any one of claims 107-116, the nucleic acid of claim 117, the vector of claim 118, or the cell of claim 119, for use in treatment of a Trk-A associated disease, preferably wherein the Trk-A associated disease is a cancer or an inflammatory disease.

123. A method of treating a Trk-A associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the protein of any one of claims 107-116 or the pharmaceutical composition of claim 120.Atorney Docket No. 45288-0513WO1124. The method of claim 123, wherein the subject is a human.

125. The method of claim 123 or 124, wherein the Trk-A associated disease is a cancer or an inflammatory disease.

126. The method of claim 125. wherein the cancer is a solid tumor or a hematological cancer.

127. The method of claim 125, wherein the inflammatory disease is osteoarthritis, juvenile chronic arthritis, or inherited arthritis.

128. A protein comprising an Interleukin- 17 (IL-17A) protein binding domain, wherein the protein comprises a sequence that is at least 80% identical to SEQ ID NO: 30, SEQ ID NO:

31. or SEQ ID NO: 32.

129. The protein of claim 128, wherein the protein comprises a sequence that is at least 85% identical to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

130. The protein of claim 128, wherein the protein comprises a sequence that is at least 90% identical to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

131. The protein of claim 128, wherein the protein comprises a sequence that is at least 95% identical to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

132. The protein of claim 128, wherein the protein comprises a sequence that is at least 99% identical to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

133. The protein of claim 128, wherein the protein comprises SEQ ID NO:

30. SEQ ID NO: 31, or SEQ ID NO: 32.

134. The protein of claim 128, wherein the protein is SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.Atorney Docket No. 45288-0513WO1135. The protein of any one of claims 128-134, wherein the protein is a single-chain protein.

136. The protein of any one of claims 128-135, wherein protein binds to IL-17A with a KD value of about 1 x 10'9M.

137. The protein of any one of claims 128-136, wherein the protein binding to IL- 17A inhibits IL-17A activity.

138. A nucleic acid comprising a sequence encoding the protein of any one of claims 128-137.

139. A vector comprising the nucleic acid of claim 138.

140. A cell comprising the nucleic acid of claim 138, or the vector of claim 139.

141. A pharmaceutical composition comprising any one of the proteins of claims 128-137, the nucleic acids of claim 138, the vector of claim 139, or the cell of claim 140; and a pharmaceutically acceptable carrier.

142. The protein of any one of claims 128-137, the nucleic acid of claim 138, the vector of claim 139, or the cell of claim 140, for use in therapy.

143. The protein of any one of claims 128-137, the nucleic acid of claim 138, the vector of claim 139, or the cell of claim 140, for use in treatment of an IL-17A associated disease, preferably wherein the IL-17A associated disease is a cancer, an infection, an autoimmune disease, or an inflammatory disease.

144. A method of treating an IL-17A associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the protein of any one of claims 128-137 or the pharmaceutical composition of claim 141.

145. The method of claim 144, wherein the subject is a human.Atorney Docket No. 45288-0513WO1146. The method of claim 144 or 145, wherein the IL-17A associated disease is a cancer, an infection, an autoimmune disease, an inflammatory disease.

147. The method of claim 146, wherein the cancer is breast cancer, colon cancer, and colorectal cancer.

148. The method of claim 146. wherein the infection is a viral infection, bacterial infection, fungal infection, protozoal infection, or parasitic infection.

149. The method of claim 146, wherein the autoimmune disease is systemic lupus erythematosus, psoriasis, rheumatoid arthritis, or multiple sclerosis.

150. The method of claim 146, wherein the inflammatory disease is acute inflammation, chronic inflammation, osteoarthritis, juvenile chronic arthritis, systemic vasculitis, systemic sclerosis, idiopathic inflammatory myopathies, sarcoidosis, idiopathic demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, non- viral hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, gluten-sensitive enteropathy, ery thema multiforme, contact dermatitis, eosinophilic pneumonia, idiopathic pulmonary fibrosis, and graft-versus-host-disease.

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