De novo designed protein binders to tumor necrosis factor superfamily receptors

Abstract

Provided are polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-73 and 99-100, wherein the polypeptide binds to a tumor necrosis superfamily receptor, fusion proteins thereof, and methods for their use.

Description

[0001]

[0002] De novo designed protein binders to tumor necrosis factor superfamily receptors

[0003] Federal Funding Statement

[0004] This invention was made with government support under Grant No. HDTRA 12110007, awarded by the Defense Threat Reduction Agency. The government has certain rights in the invention.

[0005] Sequence Listing Statement

[0006] A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on April 30, 2025 having the file name “24- 0244-WO.xml” and is 307,617 bytes in size.

[0007] Background

[0008] The design of proteins that bind with high affinity and specificity to targets of interest is a long-standing challenge in computational structural biology with applications in therapeutics, diagnostics, and beyond. To address this problem, protein design methods have generally relied on pre-existing sets of scaffolds — either native proteins or de novo designs — with well-defined tertiary structures. The advantage of using small ideal scaffolds with regular secondary structure elements and packing is that following sequence design, a reasonable fraction of designs are likely to fold as expected, but this limits the extent of shape matching achievable, particularly for targets with relatively flat surfaces lacking concavities for small miniproteins to fit into. The shape matching of binder to target has not yet reached that of highly evolved native protein-protein complexes, and previous design efforts have failed for hard targets such as those in the tumor necrosis factor receptor superfamily, including tumor necrosis factor receptor 1 (TNFR1),TNFR2, 0X40, or 4- IBB that have relatively flat and polar surfaces.

[0009] Summary

[0010] In one aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-73 and 99-100, and wherein the polypeptide binds to a tumor necrosis superfamily receptor. In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, wherein the polypeptide binds to TNFR1. In other embodiments, were in substitutions relative to the reference sequence are selected from the group consisting of SEQ ID NO: 1-8 are selected from those provided in Table 5, or substitutions relative to the reference sequence are selected from the group consisting of SEQ ID NO:9-23 and 99-100 are selected from those provided in Table 6, or substitutions relative to the reference sequence are selected from the group consisting of SEQ ID NO:24-32 are selected from those provided in Table 7.

[0011] In one embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:33-45, wherein the polypeptide binds to TNFR2. In another embodiment, the polypeptides comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:46 and 47, wherein the polypeptide binds to 0X40. In a further embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:48-73 wherein the polypeptide binds to 4-1BB. The disclosure also provides fusion proteins, comprising polypeptides of any embodiment or combination of embodiments; and one or more functional domains at the N-terminus and / or at the C- terminus of the polypeptide. In some embodiments, the one or more functional domains comprising an amino acid sequence at least at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from SEQ ID NO:74-98. In other embodiments, the fusion proteins comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 104-295. The disclosure further provides oligomers, comprising 2, 3, 4, 5, or more copies of the polypeptide or fusion protein of polypeptides of any embodiment or combination of embodiments.

[0012] The disclosure further comprises nucleic acids encoding the polypeptide or fusion protein of any embodiment herein, expression vectors comprising the nucleic acid operatively linked to a suitable control sequence, such as a promoter; host cells comprising the polypeptide, fusion protein, oligomer, nucleic acid, or expression vector of any embodiment; and pharmaceutical compositions, comprising the polypeptide, fusion protein, oligomer, nucleic acid, expression vector, and / or host cell of embodiment; and a pharmaceutically acceptable carrier.

[0013] In one aspect, the disclosure provides methods for treating an autoimmune disease, comprising administering to a subject in need thereof an amount effective to treat the disorder of the TNFR1 antagonist polypeptide of any embodiment or combination of embodiments herein, fusion proteins thereof, oligomers thereof, pharmaceutical compositions thereof, or nucleic acids or expression vectors expressing the polypeptide or fusion protein. In another aspect, the disclosure provides methods for treating cancer, comprising administering to a subject in need thereof an amount effective to treat the cancer of the TNFR2 antagonist polypeptides of any embodiment or combination of embodiments herein, fusion proteins thereof, pharmaceutical compositions thereof, or nucleic acids or expression vectors expressing the polypeptide or fusion protein. In a further aspect, the disclosure provides methods for treating an autoimmune disease, and / or promoting angiogenesis and cell proliferation in damaged tissues, comprising administering to a subject in need thereof an amount effective to treat the disorder of a TNFR2 agonist of any embodiment or combination of embodiments herein, or pharmaceutical compositions thereof. In one aspect, the disclosure provides methods for treating an autoimmune disease, comprising administering to a subject in need thereof an amount effective to treat the cancer of the polypeptide of an antagonist of 0X40 and / or 4- IBB of any embodiment or combination of embodiments herein, fusion proteins thereof, pharmaceutical compositions thereof, or nucleic acids or expression vectors expressing the polypeptide or fusion protein. In another aspect, the disclosure provides methods for treating cancer, comprising administering to a subject in need thereof an amount effective to treat the disorder of an agonist of 0X40 and / or 4- IBB of any embodiment or combination of embodiments herein, or pharmaceutical compositions thereof. The disclosure also provides in vitro methods for expanding T cells, comprising contacting a T-cell containing culture with an agonist of 0X40 and / or 4- IBB of any embodiment or combination of embodiments herein, thus expanding the T-cells.

[0014] Description of the Figures

[0015] Figure 1. Diffusion of shape complementary binding proteins. A. Previous de novo designed binders generated by docking pregenerated scaffolds bury less surface area (A2) against their target than many native complexes; the approach developed here enables the design of very large interfaces. B. TNFR1 is a challenging target, with a flat surface and few surface hydrophobic residues (PDB ID: 6KP8). Residues selected as target hotspots for RFdiffusion are shown as sticks. C. Representative RF Diffusion trajectory against TNFR1 starting from a random residue distribution placed against the target (top left). At each denoising step (Xt, top), the network generates a predicted structure (AX0, bottom), and interpolates towards this structure to generate the next step (Xt- 1). D. Comparison with previous TNFR1 design efforts. The RFdiffusion™ TNFR1 binder designs generated here have substantially higher buried SASA and contact molecular surface (CMS) than designs against TNFR1 generated previously using the Rosetta™ RIF dock method that failed to bind. Designs generated against multiple targets using RIFdock™ and short chain RFdiffusion™ are also shown for comparison. E. (top) Design models of binders TNFRl mbl and TNFRl_mb2 in complex with TNFR1 (. (bottom) SPR measurements of binding to TNFR1 (starting from 2.06 nM with a four-fold increase over 6 steps to 500 nM). F. Site saturation mutagenesis (SSM) results confirm design models of TNFR1 binders and associated entropy. All 2014 and 2033 single amino acid substitutions for TNFRl_mbl and TNFRl_mb2 were expressed on yeast surface and probed using FACS with biotinylated TNFR1 followed by deep sequencing. Positions that were strongly conserved (low entropy) were in the core and at the binding interface, while most surface residues away from the interface had high entropy). Entropy was calculated based on the overall change of affinity. Lower entropy is related to conserved interactions in the interface or core. Zoom-ins show dense interaction networks of low entropy residues.

[0016] Figure 2. Partial diffusion generates picomolar binders. A. Schematic of partial diffusion process for TNFRl_mb2. The backbone of the input structure is represented as a collection of independent residues (first panel), noise is added (second panel), and then RFdiffusion is used to remove the noise, which results in a similar but better fitting model (right) compared to the input structure. B. Partial diffusion increases interface contacts. Contact molecular surface and interface buried solvent accessible surface area is depicted for input designs and the respective partial diffused variants in squares (TNFRl_mbl), triangles (TNFRl_mb2), and circles (TNFRl_mb3). C. Partial diffusion increases interface interaction density and binding affinity. For TNFRl_mb2 an additional interface forms (left panel), while an existing interface remains largely unchanged (right). For TNFRl_mb3, improved shape matching leads to additional interactions (bottom middle insets). The corresponding TNFR1 SPR traces are on the right (6 steps of 5x dilutions from 500 nM).

[0017] Figure 3. Partial diffusion generates high-specificity TNFR2, 0X40, and 4-1BB binders. A. Phylogenetic tree of TNFSR superfamily receptors constructed using neighborjoining with branch lengths based on sequence identity. B. Comparison of structures of TNFR1, TNFR2, 0X40, and 4-1BB (PDB IDs: 7KP8, 3ALQ, 2HEV, 6BWV). C. (Top) Original design models and partial diffusion generated model for the highest affinity TNFR2, 0X40 and 4-1BB binders (also see Fig. 10). CETNFRI mbl; C2: TNFRl_mb2; C3: TNFR2_mbl; C4:OX40_mbl; C5: 4-lBB_mbl. The two TNFR1 binders were superimposed on the new targets and partially diffused to yield target-matched backbones. (Bottom) SPR measurements show that the binders are highly specific for the targets they were diffused against.

[0018] Figure 4. Design of soluble oligomeric 4-1BB and 0X40 superagonists. A.

[0019] Designed binders antagonize TNFa signaling. HEK293-Blue™ cells were incubated with 100 pM TNF-a and serial dilutions of designed binders and NFkB -dependent activation were measured. Curves are fit to data from two independent replicates. B. Schematic overview of 4-1BB signaling. Clustering of trimeric 4-1BB Ligand (4-1BB-L) with three copies of 4-1BB receptor leads to intracellular formation of TRAF1 / 2 trimers and intracellular hexamers of zinc-RING finger domains leading to downstream signaling. C. Multivalent presentation of 4- IBB binder design on cyclic oligomers activates 4- IBB signaling on luciferase reporter cell lines D. Multivalent presentation of 0X40 binder design on cyclic oligomers activates 0X40 signaling on luciferase reporter cell lines. E. Model of 4-1BB upon binding of 4-lBB_mb_l fused to the N-terminus of a cyclic hexamer compared to native complex with 4-1BB-L) (right, PDB ID: 6BWV). Distance between receptor (M101) to center is indicated (R). F. Geometry and oligomerization state dependence of designed 4- IBB and 0X40 agonists. Central panel shows maximum recorded signal at 200 pM for a series of C2-C8 oligomerbinder fusions. Dotted line indicates signal of the native ligand (0X40) or native ligand plus antibody (4- IBB). Examples of design models are shown in the surrounding panels, with fusion sites indicated by a sphere and Distance to center (R) labeled. Chains are in a gradient from N-terminus to C-terminus. Figure 5. TNFR1 binder is active in vivo. lOmg / kg of TNFRl_mb2_pdl were injected i.P. to huTNFRl knock in mice, and inflammation induced with TNFa (top) or LPS (bottom). IL-6 levels were read out 5h after injection as a measure of inflammation. Following LPS administration,, inhbition of IL-6 was compared to etanercept.

[0020] Figure 6. TNFRl_mb2_pdl variants bind to mouse ortholog. Sequence redesigned variants of TNFRl_mb2_pdl show binding to mouse orthologs of TNFR1 while retaining low picomolar affinities to the human receptors. (A) TNFRl_mb2_pdl Hl 1. (B) TNFRl_mb2_pdl Fl 1. Binding was analyzed by single cycle kinetic analysis using surface plasmon resonance (SPR). For TNFRl_mb2_pdl_Fl 1 (B), which has a disulfide stabilization, no reduction of binding capacity was observed after treatment with simulated intentstinal fluid (SIF) ( bottom panel).

[0021] Figure 7. Expression and analysis of TNFR1 binder campaign. A. 96 TNFR1 binders were expressed from e.coli and purified by size exclusion chromatography (SEC). The majority was monodispersed on SEC (upper panels) and expressed to high yields without aggregation (lower panels) B. Surface Plasmon Resonance (SPR) screening of expressed binders. All binders were tested at 4 concentrations. The left panel shows the absolute response at 2 pM binder.

[0022] Figure 8. SSM Site saturation mutagenesis of two TNFR1 binders, (A) TNFR1 mbl, and (b) TNFR1 mb2. Apparent KD in yeast display was calculated for every mutation,. Related to Fig. IF.

[0023] Figure 9. Partial diffusion is highly successful in generating specific binders. (A) TNFR1 mbl. (B) TNFR1 mb2, (C) TNFR1 mb3. 96 partially diffused TNFR1 binders were analyzed for binding to TNFR1 and related receptors by SPR. About 30% of variants derived from 3 starting backbones indicated above successfully retained or improved their binding. Binding was recorded as absolute response at 2.5 pM. In most cases, no or only weak binding to related receptors TNFR2 and 0X40 was recorded, while some variants gained reactivity to the mouse homolog of TNFR1 (mTNFRl).

[0024] Figure 10. Generation of specific binders to TNFR2, 0X40 and 4-1BB

[0025] A. 48 partially diffused binders to TNFR2 were analyzed for binding to TNFR2 and related receptors by SPR. Binding was recorded as absolute response at 2.5 pM. B. Comparison of backbones of the parental binder and the retargeted 0X40 binder OX40_mb 1. Bottom panel shows unique matching to a 0X40 specific helical turn. C. 48 4-1BB directed designs were analyzed by single cycle kinetics for 4- IBB binding on SPR. KD is indicated when curves could be fit, Rmax indicates the highest response on SPR at a concentration of 5 pM. D. AF2 model of a 4- IBB binder 4-lBB_mbl which has a unique sheet and kinked helix in the interface to match the 4-1BB fold.

[0026] Detailed Description

[0027] All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al.,

[0028] 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), “Guide to Protein Purification” in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al.

[0029] 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2ndEd. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), Dang, B. et al. SNAC-tag for sequence-specific chemical protein cleavage. Nat. Methods 16, 319-322 (2019), and the Ambion 1998 Catalog (Ambion, Austin, TX).

[0030] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0031] As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

[0032] Any N-terminal methionine residue in any polypeptide of the disclosure may be present or may be deleted. In all embodiments of the polypeptides and fusion proteins disclosed herein, 1, 2, 3, 4, or 5 residues may be deleted from the N-terminus and / or the C- terminus of the polypeptide or fusion protein while retaining activity.

[0033] All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.

[0034] Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

[0035] In a first aspect, the disclosure provides polypeptides comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-73 and 99-100, and wherein the polypeptide binds to a tumor necrosis superfamily receptor. The polypeptides of the disclosure are high affinity binders to tumor necrosis factor superfamily receptors: TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2), 0X40 (also referred to as TNFRSF4 / CD134), and 4-1BB (also referred to as TNFRSF9 / CD137).

[0036] As described in the examples, the inventors have designed de novo binding proteins for these targets, identified interface residues, and conducted site saturation mutagenesis studies to identify permissible substitutions. As further described in the examples, the polypeptides function as antagonists of their protein receptor target as monomers, and as agonists when oligomerized, and thus can be used, for example, in the methods disclosed herein. The amino acid sequence of SEQ ID NO: 1-73 and 99-100 are shown in Tables 1-4 below.

[0037] Table 1. TNFR1 binders

[0038] Table 2. TNFR2 binders

[0039] Table 3. 0X40 binders

[0040] Table 4. 4- IBB binders

[0041] In some embodiments, the polypeptides comprise an amino acid sequence at least 75% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-73 and 99-100. In other embodiments, the polypeptides comprise an amino acid sequence at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-73 and 99-100. In other embodiments, the polypeptides comprise an amino acid sequence at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-73 and 99-100.

[0042] In one embodiment, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO: 1- 32, wherein the polypeptide binds to TNFR1. In some such embodiments, the polypeptides comprise an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO: 10-18 and 25.

[0043] In some embodiments, substitutions relative to the reference sequence are selected from the group consisting of SEQ ID NO: 1-8 are selected from those provided in Table 5. In other embodiments, substitutions relative to the reference sequence are selected from the group consisting of SEQ ID NO:9-23 and 99-100 are selected from those provided in Table 6. In further embodiments, substitutions relative to the reference sequence are selected from the group consisting of SEQ ID NO:24-32 are selected from those provided in Table 7. As described in the examples, the inventors conducted site-saturation mutagenesis for the different classes of TNFR1 binders: Group G8 (SEQ ID NO: 1-8); Group F4 (SEQ ID NO:9- 23 and 99-100), and Group F12 (SEQ ID NO:24-32). Thus, in these embodiments, amino acids relative to the reference sequence include at a given residue number include the amino acids listed in the columns titled “Reference sequence residue” and “Exemplary substitutions”.

[0044] TNFR1 Binders Alternative residues

[0045] Table 5. Group G8 exemplary substitutions (relative to SEQ ID NO: 1-8)

[0046]

[0047] Table 6. Group F4 exemplary substitutions (relative to SEQ ID NO:9-23 and 99-100)

[0048]

[0049] Table 7. Group F12 exemplary substitutions (relative to SEQ ID NO:24-32)

[0050] In one embodiment, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:33-45, wherein the polypeptide binds to TNFR2. In some such embodiments, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:33-34 and 38-42.

[0051] In other embodiments, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:46 and 47, wherein the polypeptide binds to 0X40.

[0052] In further embodiments, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:48-73 wherein the polypeptide binds to 4- IBB. In some such embodiments, the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:48-57.

[0053] In some embodiments of all of polypeptide embodiments of the disclosure, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or all identified interface residues are identical (not substituted), or conservatively substituted, relative to the reference sequence. Interface residues between the polypeptide and its protein target are provided in Tables 1-4, fourth column. In other embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or all identified interface residues are identical (not substituted) relative to the reference sequence. In various further embodiments, at least 5 identified interface residues are identical relative to the reference sequence, at least 10 identified interface residues are identical relative to the reference sequence, at least 15 identified interface residues are identical relative to the reference sequence, at least 20 identified interface residues are identical relative to the reference sequence, or all identified interface residues are identical relative to the reference sequence.

[0054] In other embodiments of all of polypeptide embodiments of the disclosure, substitutions relative to the reference sequence are conservative amino acid substitutions. Such conservative amino acid substitutions involve replacing a residue by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common sidechain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0055] In another embodiment, the disclosure provides fusion proteins, comprising:

[0056] (a) a polypeptide according to any embodiment or combination of embodiments herein; and

[0057] (b) one or more functional domains at the N-terminus and / or at the C-terminus of the polypeptide.

[0058] In these embodiments, any functional domain may be fused to the polypeptides of the disclosure. In various non-limiting embodiments, the functional domain may comprise, for example, a targeting domain, a detectable domain, a scaffold domain, a secretion signal, a purification tag, an Fc domain, an oligomerization domain, a domain for half-life extension (e.g. albumin binding domain), or a further therapeutic peptide domain.

[0059] In another embodiment, the polypeptides or fusion proteins bind their target with nanomolar or picomolar affinity or better, as disclosed in the examples.

[0060] In another embodiment, the disclosure provides oligomers, comprising 2, 3, 4, 5, or more copies of the polypeptides or fusion proteins of any embodiment or combination of embodiments herein. The oligomers may comprise conjugation of the polypeptides or fusion protein to any domain capable of multimerization. In some embodiments, the polypeptides or fusion proteins are fused to an oligomerization domain, wherein the oligomer comprises 2, 3, 4, 5, or more copies of the oligomerization domain-containing fusion protein. In these embodiments, the oligomerization domain may be present N-terminal or C-terminal to the polypeptide of the first aspect of the disclosure. In all embodiments of fusion proteins, the polypeptides may be directly fused to the one or ore functional domains, or may be linked by an amino acid linker of any suitable length and amino acid composition.

[0061] Such fusion proteins include but are not limited to C- or N-terminal fusion to a polypeptide listed in Table 8, SEQ ID NO:74-98, where any N-terminal methionine residue is optional and may be present or deleted.

[0062] Table 8

[0063] In these embodiments, the fusion proteins act as agonists of their target, and can be used in the methods of the disclosure. In non-limiting embodiments, the fusion proteins comprise or consist of an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 104-295. The amino acid sequences of these fusion proteins are provided in Tables 9-10.

[0064] Table 9

[0065] Table 10

[0066] In another aspect, the disclosure provides nucleic acids encoding the polypeptide or fusion protein of any embodiment or combination of embodiments of the disclosure. The nucleic acid sequence may comprise single stranded or double stranded RNA or DNA in genomic or cDNA form, or DNA-RNA hybrids, each of which may include chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Such nucleic acid sequences may comprise additional sequences useful for promoting expression and / or purification of the encoded peptide or chimeric molecular construct, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptide or fusion protein of the disclosure.

[0067] In a further aspect, the disclosure provides expression vectors comprising the nucleic acid of any aspect of the disclosure operatively linked to a suitable control sequence, such as a promoter. “Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the disclosure are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector.

[0068] In another aspect, the disclosure provides host cells that comprise the polypeptide, fusion protein nucleic acid or expression vector (i.e.: episomal or chromosomally integrated) disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of the disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate coprecipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.

[0069] The disclosure further comprises pharmaceutical compositions, comprising:

[0070] (a) the polypeptide, fusion protein, oligomer, nucleic acid, expression vector, and / or host cell of any embodiment or combination of embodiments herein; and

[0071] (b) a pharmaceutically acceptable carrier. The compositions may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and / or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the composition includes a bulking agent, like glycine. In yet other embodiments, the composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate- 85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the composition additionally includes a stabilizer, e.g., a molecule which substantially prevents or reduces chemical and / or physical instability of the nanostructure, in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0072] The polypeptide, fusion protein, nucleic acid, expression vector, and / or host cell may be the sole active agent in the composition, or the composition may further comprise one or more other agents suitable for an intended use.

[0073] Blocking pro-inflammatory effects of TNF-alpha, for treatment of immune diseases like rheumatoid arthritis, psoriasis, and inflammatory bowel disease, as well as neurodegenerative diseases where inflammation is a contributing factor (e.g. Alzheimers, multiple sclerosis). In particular, treatment of inflammatory bowel disease is of interest, due to the potential of highly stable mini proteins for oral administration. Thus, in another aspect, the disclosure provides methods for treating an autoimmune disease, including but not limited to rheumatoid arthritis, psoriasis, and inflammatory bowel disease, and / or a neurodegenerative disease where inflammation is a contributing factor (including but not limited to Alzheimer’s disease and multiple sclerosis), comprising administering to a subject in need thereof an amount effective to treat the disorder of the TNFR1 antagonist polypeptide of any embodiment or combination of embodiments herein, fusion proteins thereof, oligomers thereof, pharmaceutical compositions thereof, or nucleic acids or expression vectors expressing the polypeptide or fusion protein.

[0074] Several cancers use the TNFR2 pathway to promote tumor growth and suppress the immune response against the tumor. Antagonistic minibinders can thus be explored for treatment of such cancers (e.g. melanoma, ovarian cancer etc.). Thus, in one aspect, the disclosure provides methods for treating cancer, including but not limited to a melanoma or ovarian cancer, comprising administering to a subject in need thereof an amount effective to treat the cancer of the TNFR2 antagonist polypeptides of any embodiment or combination of embodiments herein, fusion proteins thereof, pharmaceutical compositions thereof, or nucleic acids or expression vectors expressing the polypeptide or fusion protein.

[0075] Activating TNFR2 promotes survival of Tregs and can thus be applied for treatment of inflammatory disease described above, alone or in combination with TNFR1 antagonists. TNFR2 agonists can enhance tissue regeneration by promoting angiogenesis and cell proliferation in damaged tissues. Activation of TNFR2 has shown potential in promoting neuronal survival and the methods can thus be used for treatment of neurodegenerative diseases. Thus, in a further aspect, the disclosure provides methods for treating an autoimmune disease, including but not limited to rheumatoid arthritis, psoriasis, and inflammatory bowel disease, and / or neurodegenerative disease (including but not limited to Alzheimer’s disease and multiple sclerosis), and / or promoting angiogenesis and cell proliferation in damaged tissues, comprising administering to a subject in need thereof an amount effective to treat the disorder of a TNFR2 agonist of any embodiment or combination of embodiments herein, or pharmaceutical compositions thereof. In one embodiment, the method further comprises administering the subject a TNFR1 antagonist polypeptide of any embodiment or combination of embodiments herein, fusion proteins thereof, oligomers thereof, pharmaceutical compositions thereof, or nucleic acids or expression vectors expressing the polypeptide or fusion protein.

[0076] Antagonists of 0X40 and / or 4- IBB can be used, for example, controlling excessive immune responses in autoimmune diseases and graft vs host disease, reducing activity of autoreactive T-cells. Thus, the disclosure also provides methods for treating an autoimmune disease, including but not limited to rheumatoid arthritis, psoriasis, and inflammatory bowel disease, and / or graft versus host disease, comprising administering to a subject in need thereof an amount effective to treat the cancer of the polypeptide of an antagonist of 0X40 and / or 4- IBB of any embodiment or combination of embodiments herein, fusion proteins thereof, pharmaceutical compositions thereof, or nucleic acids or expression vectors expressing the polypeptide or fusion protein.

[0077] Agonists of 0X40 and / or 4-1BB can be used, for example, to stimulate T cells, enhancing proliferation, survival, and memory'. This is relevant both for in vitro expansion, but also as a treatment of various cancers by activating cytotoxic T-cells, and as costimulatory molecules for CAR T-cell therapy. Thus, the disclosure further provides methods for treating cancer, comprising administering to a subject in need thereof an amount effective to treat the disorder of an agonist of 0X40 and / or 4- IBB of any embodiment or combination of embodiments herein, or pharmaceutical compositions thereof. The disclosure also provides in vitro methods for expanding T cells, comprising contacting a T-cell containing culture with an agonist of 0X40 and / or 4-1BB of any embodiment or combination of embodiments herein, thus expanding the T-cells.

[0078] As used herein, "treat" or "treating" a disorder means accomplishing one or more of the following in a subject with the disorder: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).

[0079] The subject may be any subject that has a relevant disorder or is / may be at risk of the relevant disorder. In one embodiment, the subject is a mammal, including but not limited to humans, dogs, cats, horses, cattle, etc.

[0080] As used herein, an “effective” amount refers to an amount of the polypeptide, fusion protein, oligomer, nucleic acid, expression vector, and / or host cell that is effective for treating the disorder. The polypeptides, fusion proteins, oligomers, nucleic acids, expression vectors, and / or host cells are typically formulated as a pharmaceutical composition, such as those disclosed above, and can be administered via any suitable route, including but not limited to orally, by inhalation spray, ocularly, intravenously, subcutaneously, intraperitoneally, and intravesicularly in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0081] Any suitable dosage range may be used as determined by attending medical personnel. Dosage regimens can be adjusted to provide the optimum desired response. A suitable dosage range for the polypeptides or fusion proteins may, for instance, be 0.1 ug / kg- 100 mg / kg body weight; alternatively, it may be 0.5 ug / kg to 50 mg / kg; 1 ug / kg to 25 mg / kg, or 5 ug / kg to 10 mg / kg body weight. In some embodiments, the recommended dose could be lower than 0.1 mcg / kg, especially if administered locally (such as by intra-tumoral injection). In other embodiments, the recommended dose could be based on weight / m2 (i.e. body surface area), and / or it could be administered at a fixed dose (e.g., .05-100 mg). The polypeptides, fusion proteins, nucleic acids, expression vectors, and / or host cells can be delivered in a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or more times) as determined by an attending physician.

[0082] The polypeptides, fusion proteins, oligomers, nucleic acids, expression vectors, and / or host cells made be administered as the sole therapeutic agent, or may be administered together with (i.e.: combined or separately) one or more other therapeutic agents,

[0083] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize.

[0084] Examples

[0085] The design of proteins that bind with high affinity and specificity to targets of interest is a long-standing challenge in computational structural biology with applications in therapeutics, diagnostics, and beyond. To address this problem, protein design methods have generally relied on pre-existing sets of scaffolds — either native proteins or de novo designs — with well defined tertiary structures. The advantage of using small ideal scaffolds with regular secondary structure elements and packing is that following sequence design, a reasonable fraction of designs are likely to fold as expected, but this limits the extent of shape matching achievable, particularly for targets with relatively flat surfaces lacking concavities for small miniproteins to fit into. Indeed, the contact molecular surface (CMS) of de novo- designed binding proteins to date is lower than that of many native protein complexes (Fig. 1A).

[0086] We reasoned that the limited shape matching achievable by scaffold dependent and / or short chain approaches could be overcome by using RFdiffusion™ to directly generate larger proteins starting from completely random residue distributions in the presence of the target of interest without any guidance from pre-existing scaffolds. Completely unconstrained RFdiffusion™ can generate a wide diversity of folds and assemblies, and proteins with folds that wrap around extended helical peptides. We set out to explore whether RFdiffusion™ trajectories conditioned only on the structure of a folded protein target, and not biased towards any particular scaffold or limited by the number of available residues, could generate folds with shapes matched to the target that extend over a large portion of the surface, and whether such an approach could enable the generation of high-affinity binders to targets for which previous computational design efforts had failed.

[0087] Binder design

[0088] We chose to focus on the tumor necrosis factor receptor superfamily (TNFRSFs), which includes many important drug targets, including the TNF receptor 1 (TNFR1), which plays a key role in inflammatory disease (6). Like other family members, TNFR has an extended flat and largely polar surface lacking the concave sites with several hydrophobic residues that previous de novo design efforts have successfully targeted (Fig. IB) — indeed multiple attempts to generate binders to TNFR and other family members using the approach of Cao et al. (7) met with little success (unpublished). We first attempted to use RFdiffusion™ as described in Watson et al. (7, 4) to generate binders to TNFR1, using guidance from 65 aa scaffold libraries or limited to chains less than 65 residues but met with little success.

[0089] We next set out to adapt RFdiffusion™ to generate backbones with a more extended contact surface that can engage dispersed surface hydrophobic residues (Fig. IB). Protein interactions, like protein folding, are largely driven by hydrophobic interactions, and targets with only a few surface hydrophobic residues distant in space have been particularly challenging. On TNFR, the few surface hydrophobic residues are separated by distances of up to 28 A — too small for 65 residue proteins to simultaneously engage. To overcome this limitation of previous approaches, random Gaussian residue clouds of up to 120 residues were placed at the native ligand (TNF) interface, and RFdiffusion™ was biased to form contacts with these dispersed hydrophobic residues. This generated a variety of unique backbones quite different from those in previous campaigns, but complementary in shape to TNFR1. ProteinMPNN™ was used to design sequences for these backbones in complex with TNFR1 to favor both folding to the intended structure and binding to the target. To further sample around promising designs predicted by AlphaFold2™ (AF2) to form complexes (pae_interaction <20), we extracted additional backbones from preceding diffusion timesteps, as the predicted structure was already close to the final structure at around timestep 120 out of 200 (Fig. 1C). The designs predicted to most strongly bind the TNFR1 (interface pae < 7.5) and fold to the target backbone (pLDDT >85) were selected for experimental characterization. The contact molecular surface (CMS) and buried solvent accessible surface area (SASA) of these designs was substantially higher than designs from previous design campaigns (Fig. ID).

[0090] Genes encoding the 96 designs were obtained, and the proteins expressed in E coli. Despite the less regular structures and longer lengths than most previous binder designs, 90 of 96 expressed well and were primarily monomeric (Fig. 7A). Six of the designs bound to TNFR1 in surface plasmon resonance (SPR) experiments (Fig. 7B), with designs TNFRl_mbl and TNFRl_mb2 having KDS of 12 nM and 8 nM respectively. Both designs were highly specific, with no detectable binding to TNFR2. The tertiary structure was quite distinct from previously designed de novo binders, and the designs interact with TNFR1 over an extended region (Fig. ID). Design TNFRl_mb2 has an unusual V shaped fold with a very high contact molecular surface (CMS) for TNFR of 795 A2, substantially more than the average 490 A2of previous diffused designs (4). Design TNFRl_mbl also has a binding mode less regular than that of previous de novo designed binders with a connecting loop inserted in the TNFR1 binding cleft, and a similarly high CMS of 897 A2. For both designs, we obtained a high resolution binding footprint by determining the effect on binding of every amino acid substitution at every position one at a time (4047 substitutions in total) (Fig. 8). The results were closely consistent with the design models, with substitutions impacting binding concentrated at the designed interface and in the protein core where they would disrupt folding. The most conserved interactions for both designs centered around the hydrophobic patches involving TNFR1 residues 107 / 111 and residues 38 / 40 (Fig. IE) spanned by more polar interactions in the center of the interface. Taken together, the close agreement of the site saturation mutagenesis (SSM) footprints with the design model interfaces, and AF2 and RoseTTAFold2™ (RF2) complex predictions with the designed structures (pAE interface of 5. 1 and 4.08, CA RMSD to design of 0.6 and 0.97 for designs TNFRl mbl and TNFRl_mb2 respectively, using AlphaFold2™-multimer model 1), suggest that both TNFRl mbl and TNFRl_mb2 bind to TNFR1 as designed.

[0091] Tumor necrosis factor (TNF-a) is a trimer that binds TNFR1 with high (19 pM) affinity (7), and to effectively outcompete this interaction with a monomeric protein to counter inflammation even higher binding affinities are required. To further optimize the affinity of the TNFRl_mb2 and TNFRl mbl designs, and the TNFRl_mb3 design, which also bound specifically, rather than combining beneficial substitutions from the SSM libraries, which requires considerable experimental screening (7), we used partial diffusion (Fig. 2A): backbones were partially noised (over 15 - 25 steps; 50 steps yield a completely random distribution), followed by RFdiffusion™ denoising which yielded new backbones resembling but distinct from the original designs (RMSD 0.58 to 4.96). We generated 25,000 partially diffused backbones around each starting structure, and following ProteinMPNN™, the 32 designs for each starting structure which AF2 most confidently predicted bound TNFR1 in the designed binding mode (pae_interaction < 5) were selected for experimental characterization. These partially diffused designs had substantially greater CMS and buried SASA than the parent designs in all three cases (Fig. 2B). The designs were expressed in E.coli, and TNFR binding was measured by SPR. Most (94 out of 96) of the designs were expressed at high levels, and 30% (28 out of the 94 expressed designs) bound TNFR1 (Fig. 9). Partial diffusion increased the binding affinity of TNFRl_mb2 by three orders of magnitude to 9 pM, while TNFRl_mb3 affinity increased from weak binding in the uM range to 20 nM (Fig. 2C). Improvements were smaller for the more regular TNFRl_mbl backbone; free diffusion sampling appears to have already found a close-to-ideal solution for this binding mode. For TNFRl_mb2, the considerable increase in affinity brought about by partial diffusion likely reflects an additional interface and an overall better fit to the binding cleft with several additional contacts (Fig. 2C). The 9 pm affinity of the partially diffused TNFRl_mb2, which we refer to as TNFRl_mb2_pdl below, is considerably higher than any previously described monomeric TNFR1 binding protein.

[0092] Given the success of partial diffusion in increasing binding affinities, we investigated whether a similar approach could be used to switch specificity to other TNFR family members, which are diverse in sequence, but have a very similar overall fold (Fig. 3 A and B). We placed binders TNFRl_mb2, TNFRl mbl, and TNFRl_mb3 on TNFR superfamily members TNFR2, 0X40, and 4- IBB (by superimposing the latter receptors on the TNFR1 in the design models), and carried out 25,000 noising and design trajectories for each combination consisting of the addition of random Gaussian noise, RFdiffusion™ denoising, and ProteinMPNN™ sequence design. For TNFR2, 1323 designs had AF2 complex predictions with pae_interaction <7.5 — a substantially higher success rate than achieved by free diffusion on TNFR1. For each of the receptors, 48 designs were experimentally characterized. For TNFR2, 32% of the designs bound with high specificity (Fig. 10A); the highest affinity design had a KD of 52 pM to TNFR2 and no affinity for the other family members tested. Unlike TNFR2, which shares a common ligand with TNFR1 (TNF-a), 0X40 has a different ligand and thus a more distinct binding interface; despite this difference in natural ligand partial diffusion starting from the TNFR1 binders yielded an 0X40 binder with a KD of 24 nM. As expected, this binder was shifted more substantially both in approach angle and tertiary structure compared to the parental design than the TNFR2 binders (Fig. 10S6B). For the even less related 4- IBB, an additional round of partial diffusion was required to achieve a pae_interaction less than 7, but the experimental success rate was still high, with 22 out of 48 tested designs binding specifically to their target, with the highest affinities of 60 and 64 nM. Design 4-lBB_mbl illustrates how partial diffusion can conform backbones to a target, in this case, by introducing an unusual kinked helix and a short betasheet to pair with the unique receptor fold (Fig. 10D).

[0093] The picomolar affinity of the TNFR1 binders makes them possible candidates for blocking inflammation. To date, targeting of the TNF-a pathway has primarily focused on binding to circulating TNF-a due to the fact that antibodies targeting TNFR1, owing to their bivalency, can activate, rather than suppress, TNF-a signaling. We investigated if our (monomeric) designs could inhibit TNF-a signaling on a TNF-a HEK293 reporter cell line (InvivoGen), which monitors TNF-a signaling via AP-1 / NF-KB dependent activation of a SEAP reporter gene (Fig. 4A). We found that the designs potently inhibited TNF-a signaling, with an EC50 for the best design at 43 pM.

[0094] TNFRl_mb2_pdl was tested for capacity to inhibit TNFa induced inflammation in a hTNFRIKI mouse model. At a dose of 5 mg / kg i.p., the antagonist efficiently blocked TNFR1 and reduced inflammation (measured by serum IL-6 levels) to baseline levels, both after direct induction of inflammation with TNFa, as well as indirect with LPS. Importantly, binder alone did not induce significant inflammatory effects by unwanted receptor clustering. Moreover, TNFRl_mb2_pdl showed significantly better reduction of IL-6 levels 5h after LPS induced inflammation compared to lOmg / kg s.c. etanercept. (Fig. 5)

[0095] We further engineered the designs for stability and mouse receptor binding by redesigning the sequence using ProteinMPNN™ and introducing cysteines. Two variants (SEQ ID NO:99 and 100) exhbitied enhanced binding to the mouse ortholog, and variant TNFRl_mbs_pdl_Fl l was highly resistant to treatment with simulated intestinal fluid (SIF). (Fig. 6)

[0096] Agonist design

[0097] For 0X40 and 4-1BB, which have been widely studied for expanding T cells for cancer treatment, agonists could have therapeutic potential. Unlike TNFa / TNFRl, 4-1BB signaling is not activated by soluble trimeric ligands alone-the physiological ligands are in the plasma membrane of adjacent cells, and induce 4- IBB arrangements with longer range order (Fig. 4B). Signaling has been achieved using antibody-generated ligand networks to drive higher-order complexes (8-10), but while such ligands are soluble, they are quite heterogeneous. To explore the generation of well-defined monodisperse 4- IBB agonists, we fused the 4- IBB binder to designed homo-oligomers with different valencies and spacings between fusion sites (dots in Fig. 4). See Tables 9-10. We found that monomers and C2 or C3 oligomers did not signal, consistent with the lack of signaling of native trimeric ligands. In contrast, signaling was observed with C4, C5, C6, and C8 oligomers (Fig. 4C). The strongest signal was observed for a C6 construct which arranges the receptors at similar distances as the native ligand, but with higher valency (Fig. 4E). Over a set of 44 oligomers (Fig. 4F), valency was the strongest determinant of signal strength, with no signal for C1-C3, a consistent but low signal for C4, and a higher signal for C5 and C6. Beyond valency, the geometry of association also influenced the extent of signaling, as illustrated by the structure schematics in Fig. 4F: higher order oligomers that separate the receptors by distances significantly greater than the native ligand exhibited low or no signal as did those that would likely clash with the receptor or penetrate the membrane (Fig. 4F). Overall, the requirement for higher order valencies is consistent with the proposed signaling mechanism inferred from the intracellular hexameric arrangement (11) (Fig. 4B), but it remains to be determined how bringing in just one more subunit (in the C4 case) leads to agonism.

[0098] For 0X40, we again tested a range of different oligomeric states, and observed a quite different pattern. In contrast to 4-1BB, trimeric constructs were effective agonists, consistent with the fact that 0X40 can be activated by soluble trimeric ligands. Monomeric and dimeric binder constructs did not signal, while oligomeric constructs with three, four, or five binding modules efficiently activated signaling (Fig. 4D,F).

[0099] For both 4- IBB and 0X40, both the EC50 and Emax (maximum signal) varied considerably among the oligomeric constructs, indicating a substantial opportunity for finetuning the response by modulating valency and geometry, both to investigate the mechanism of signaling through this important class of receptors, and for therapeutic applications. Particularly interesting are the substantially higher Emax of the best 0X40 and 4-1BB synthetic agonists compared to the native ligand in the 0X40 case and antibody-ligand assemblies in the 4-1BB case; these could be particularly useful for expanding T-cell populations.

[0100] Conclusions For therapeutic challenges for which antagonism without any risk of agonism is necessary, high-affinity monomeric binders could have advantages over bivalent antibodies, which can potentially dimerize the target receptor and activate signaling. TNF-a and TNFR are key drug targets given the central role this interaction plays in inflammatory disease; current therapies primarily target the ligand TNF-a instead of TNFR1 to avoid potential activation of inflammatory responses, but binding TNF-a also inhibits potentially antiinflammatory signaling through TNFR2, which could contribute to unwanted side effects of this important class of drugs (13). The very large interfaces of the TNFR1 binders, even larger than the native interface despite being only 107 amino acids, could likely enable even higher affinity antagonism into the fM range, and the high stability and likely low cost of production of small designed proteins could enable oral administration for gut disease. As illustrated by our 4- IBB and 0X40 superagonists, the high affinity monomeric binders enable the construction of a wide variety of soluble signaling molecules, offering far more control than current native ligand plus crosslinking antibody-based approaches.

[0101] More generally, the ability to generate high affinity and specificity binders to therapeutically important and structurally challenging protein targets without having to immunize animals, screen large random libraries, or test thousands of design candidates ushers in a new era for binder design and therapeutic candidate discovery. The number of sequences tested (96 free diffusion and 96 partial diffusion designs for TNFR1, and 48 partial diffusion designs for TNFR2, 0X40, and 4- IBB) is far fewer than in previous studies in which libraries of tens of thousands of designs were screened using yeast display, and no random or experimentally guided optimization was involved other than selecting the best of the 96 first round designs for partial diffusion. The 9 pM affinity for TNFR1 and 52 pM for TNFR2 are the highest we are aware of for monomeric binders to these targets; for comparison, the antibodies that likely bind bivalently have affinities of up to 680 pM, while monomeric Fabs, scFvs, and nanobodies are in the range of 10-100 nM (12). This high affinity for targets for which multiple previous binder design efforts failed likely reflects the very high designed shape complementarity and buried surface area. Indeed, the amount of surface area these designs bury on their targets is substantially higher than in previous minibinder design efforts, and the buried surface area per residue rivals that of the native complexes, which have evolved over hundreds of millions of years (Fig. 1A). The combination of RFdiffusion™ starting from random residue clouds of >100 amino acids and partial diffusion to optimize affinity and achieve high specificity for family members provides a very powerful and general approach to obtaining high potency and affinity binders to challenging classes of targets.

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[0124] 15. B. I. M. Wicky, L. F. Milles, A. Courbet, R. J. Ragotte, J. Dauparas, E. Kinfu, S. Tipps, R. D. Kibler, M. Baek, F. DiMaio, X. Li, L. Carter, A. Kang, H. Nguyen, A. K. Bera, D. Baker, Hallucinating symmetric protein assemblies. Science 378, 56-61 (2022). 16. N. I. Edman, R. L. Redler, A. Phal, T. Schlichthaerle, S. R. Srivatsan, A. Etemadi, S. J. An, A. Favor, D. Ehnes, Z. Li, F. Praetorius, M. Gordon, W. Yang, B. Coventry, D. R. Hicks, L. Cao, N. Bethel, P. Heine, A. Murray, S. Gerben, L. Carter, M. Miranda, B. Negahdari, S. Lee, C. Trapnell, L. Stewart, D. C. Ekiert, J. Schlessinger, J. Shendure, G. Bhabha, H. Ruohola-Baker, D. Baker, Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies, bioRxiv (2023)p. 2023.03.14.532666.

[0125] Supplementary Materials and Methods

[0126] Design of protein binders to TNFRSFs

[0127] To design de novo binders to TNFRSFs using RFdiffusion™, we initially generated backbones to TNFR1 based on a cryo-EM structure (PDB ID: 7KP8). We generated 30,000 initial designs using RFdiffusion™ (- / ). We targeted binders using input “hotspot” residues to a specific site on the target protein. The hotspots selected were as follows (chain and residue index from PDB): F67, F70, F95, Fl 12.

[0128] In line with current best practice, we used the ProteinMPNN™-FastRelax™ protocol described in Bennett et al (14), this protocol starts with a round of ProteinMPNN and then cycles between FastRelax™ and ProteinMPNN™ to attempt to iteratively improve the sequence and structure agreement. Two sequences per design were generated. To filter designs we ran AF2 with an initial guess and target templating. Briefly, this configuration of AF2 runs without a multiple sequence alignment and without template information for the de novo binder, which ensures that predictions are not biased towards examples that have sequence or structural homology to the PDB. This configuration uses the template feature in AF2 to provide the exact structure of the target protein, as we are designing with a rigid target and know the structure of the target a priori, we desire for AF2 to keep the target fixed and only predict the dock and structure of the de novo binder. Finally, this configuration of AF2 initializes the dock and structure of the de novo binder with the RFdiffusion™ design model of the dock and structure.

[0129] For designs that were predicted as complex with pae_interaction <20, multiple backbones were extracted from the trajectories (10 backbones at every 5thtimestep from 0- 50), and the ProteinMPNN™-FastRelax™-AF2 pipeline repeated, to diversify designs and achieve a sufficient number to order on a 96 scale. Partial diffusion

[0130] RFdiffusion™ was modified to allow the input structure to be noised only up to a user-specified time step instead of completing the full noising schedule as described previously (5). The starting point of the denoising trajectory is therefore not a random distribution, but contains information about the input distribution resulting in denoised structures that are structurally similar to the input. The AF2 models of the initial binders TNFRl_mbl-3 were used as inputs to partial diffusion, either directly or after superimposing and replacing the receptor with TNFR2, 0X40, or 4- IBB (PDB IDs: 3ALQ, 2HEV, 6BWV respectively). The models were subjected to 15 or 25 noising time steps out of a total of 50 time steps in the noising schedule, and subsequently denoised. An auxiliary potential to promote interface contacts was used in some cases. Approximately 20,000 partially diffused designs were generated for each target. The backbones in the resulting library were sequence designed as described above for free diffusion designs. The designs with the lowest pAE interaction and pLDDT >85 were selected for experimental characterization.

[0131] Plasmid construction

[0132] Protein binder designs were ordered as synthetic genes (eBlocks™, Integrated DNA Technologies) with compatible Bsal overhangs to the target cloning vector, LM0627 (15) for Golden Gate assembly. LM0627 is a modified expression vector containing a Kanamycin resistance gene and a ccdb lethal gene between Bsal cut sites. Subcloning into LM0627 results in the following product: MSG-[protein]- GSGSHHWGSTHHHHHH (SEQ ID NO: 101), with the C-terminal SNAC cleavage tag and 6XHis affinity tag respectively.

[0133] Oligomerized binders were generated by cloning those eBlocks™ in similar vectors which resulted in both C- and N- terminal fusion to 24 oligomerization domains, and in a final construct of MSG-[binder]-GSGS-[oligomerization domain]- GSGSHHWGSTHHHHHH (SEQ ID NO: 102) or [oligomerization domain] -GSGS- [binder] - GSGSHHWGSTHHHHHH (SEQ ID NO: 103). Oligomerization domains were described previously (15, 16), a full list of oligomerization domains used can be found in Table 8, minus the linker domains and optional N- and C-terminal residues.

[0134] Protein expression and purification

[0135] For protein binder expression screens, a previously reported protocol (15) was followed with some modifications as denoted. In short, subcloning reactions of designs were carried out in 96-well PCR plates in 1 pL volume. Reaction mixtures were then transformed into a chemically competent expression strain (BL21(DE3)), and 1-hour outgrowths were split directly into four 96-deep well plates containing 1 mL of auto-induction media (autoclaved TBII media supplemented with Kanamycin, 2 mM MgSO4, IX 5052) for a final total volume of approximately 4 mL. The following day (20-24 hrs later), cells were harvested and lysed, and clarified lysates were applied directly to a 100 pL bed of Ni-NTA agarose resin in a 96-well fritted plate equilibrated with a Tris wash buffer. After sample application and flow through, the resin was thoroughly washed, and samples were eluted in 200 pL of a Tris elution buffer containing 500 mM imidazole.

[0136] All eluates were sterile-filtered with a 96-well 0.22 pm filter plate (Agilent 203940- 100) prior to size exclusion chromatography (SEC). Protein designs were then screened via SEC using an AKTA™ FPLC outfitted with an autosampler capable of running samples from a 96-well source plate. Protein binders were run on a Superdex™75 Increase 5 / 150 GL column (Cytiva 29148722), and symmetric oligomers were run on a Superdex™200 Increase 5 / 150 GL column (Cytiva 28990945). For all binding proteins, HBS-EP+ ( 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20) was used as a running buffer for subsequent SPR analysis without buffer exchange. For oligomerized binders, 20 mM NaPhos pH 7.4, and 100 mM NaCl was used as a running buffer, 0.25 mL fractions were collected from each run, and selected fractions were pooled for further analysis.

[0137] For larger scale protein purification, protein expression was performed using 50 mL of auto-induction media (autoclaved TBII media supplemented with Kanamycin, 2mM MgSO4, IX 5052), and grown for 24h at 37°C. The cells were harvested by spinning at 4,000 x g for 10 min and then resuspended in lysis buffer (100 mM Tris-HCl, 200 mM NaCl, 50 mM imidazole). Then, the cells were lysed by sonication in a Qsonica™, Q500 with a 4- pronged horn for 2:30 min ON total, with an amplitude of 80%. Soluble fractions were clarified by centrifugation at 4,000 x g for 30 minutes, and were subsequently purified by affinity chromatography using bed Ni-NTA resin (Qiagen or Thermo Fisher) on a vacuum manifold. A series of 3 washes using wash buffer (20 mM Tris-HCl, 200 mM NaCl, 50 mM imidazole) was performed prior to elution with elution buffer (20 mM Tris-HCl, 200 mM NaCl, 500 mM imidazole). After elution, protein samples were filtered and injected into an autosampler-equipped Akta pure system on a Superdex™ S75 Increase 10 / 300 GL column (Cytiva 28-9909-44) at room temperature. The SEC running buffer was 20mM Tris-HCl, lOOmM NaCl pH 8.

[0138] Surface Plasmon Resonance Binding kinetics were analyzed via Surface Plasmon Resonance (SPR) on a Biacore™ 8K (Cytiva). Binding to various TNFRSFs was measured by capturing Fc-tagged receptor ectodomain of TNFR1, TNFR2, 0X40, or 4- IBB (all Sino Biological, #10872- H02H-100, #10417-H03H, #10481-H02H, #10041-H02H) on a Protein A chip (Cytiva #29127556), by injecting 0. 125 pg / mL receptor at a flow rate of 10 pL / min in HBS-EP+ (0.01 M HEPES pH 7.4, 0. 15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20, Cytiva #BR100669) aiming for a capture level of -250 response units. Analytes were diluted in HBS-EP+ and injected at a flow rate of 30 pL / min to monitor association. HBS-EP+ was used as a running buffer during dissociation at a flow of 30 pL / min. Screens were performed at a single dilution, full kinetics by running single cycle kinetics, injecting increasing concentrations of ligand. Association and dissociation times and concentration ranges were varied to suit the respective analytes. Binding kinetics were determined by fitting curves assuming a 1: 1 Langmuir interaction, using the Cytiva evaluation software.

[0139] Yeast surface display

[0140] Saccharomyces cerevisiae EBY 100 strain cultures were grown in C-Trp-Ura medium supplemented with 2% (w / v) glucose. For induction of expression, yeast cells were centrifuged at 6,000g for 1 min and resuspended in medium supplemented with 0.2% (w / v) glucose at the cell density of 1 x 107cells per mb and induced at 30 °C for 16-24 h. Cells were washed with PBSF (PBS with 1% (w / v) BSA) and labeled with biotinylated TNFR1 (R&D Systems). The cells were first incubated with biotinylated targets, washed and secondarily labeled with anti-c-Myc fluorescein isothiocyanate (FITC, Miltenyi Biotech) and streptavidin-phycoerythrin (SAPE, ThermoFisher). For SSM libraries, two rounds of sorts were applied and in the third round of screening, the libraries were titrated with a series of decreasing concentrations of targets to enrich mutants with beneficial mutations.

[0141] TNF-a signaling assays

[0142] HEK-Blue™-TNF-a cells (Invivogen) were cultured in DMEM media and maintained according to standard protocols. Binders were serially diluted sevenfold from a starting concentration of 1 pM in Dulbecco's Modified Eagle Medium (DMEM) media. Equal volumes of the prepared binders were added to 96-well plates at concentrations ranging from 1 pM to 0.008 pM. Subsequently, HEK-Blue™TNF-a cells were added to each well at a density of 50,000 cells per well and incubated with the binders for 10 minutes. After the incubation period, TNF-a at a final concentration of 0.01 nM was added to each well to compete with the binders for binding to TNF-a receptors on the HEK-Blue™-TNF-a cells. Plates were then incubated at 37°C for 24 hours to allow for TNF-a-induced activation of signaling pathways. Following incubation, the cell supernatant was collected, and 10 pL of the supernatant was mixed with 90 pL of QuantiBlue™ reagent (Invivogen #rep-qbs3). The mixture was incubated at 37°C for 30 minutes to allow for the conversion of the reagent, and the optical density at 615 nm (OD615) was measured using a Neo2 plate reader.

[0143] OX40 / 4-1BB signaling assays

[0144] For 0X40 and 4- IBB signaling assays, commercially reporter Jurkat cells assay kits were used. (Promega) In both cases, cells were thawed, and 20,000 cells were distributed in 25 pL RPMI media supplemented with 10% FCS to 384-well plates. An equal amount of diluted binder oligomers was added to cells in RPMI media supplemented with 10% FCS. After 8h of incubation at 37°C, cells were lysed with lysis buffer supplemented with BrightGlo substrate as supplied by the manufacturer, and transferred to black 384-well plates. After 10 min of incubation, luminescence was read out with a Neo2 plate reader.

Claims

We claim1. A polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-73 and 99-100, and wherein the polypeptide binds to a tumor necrosis superfamily receptor.

2. The polypeptide of claim 1 comprising an amino acid sequence at least 75% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-73 and 99- 100.

3. The polypeptide of claim 1 comprising an amino acid sequence at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-73 and 99- 100.

4. The polypeptide of claim 1 comprising an amino acid sequence at least 95% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 1-73 and 99- 100.

5. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, wherein the polypeptide binds to TNFR1.

6. The polypeptide of claim 5, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO : 10- 18 and 25.

7. The polypeptide of claim 5 or 6 comprising an amino acid sequence at least 75% identical to the amino acid sequence of the reference sequence.

8. The polypeptide of claim 5 or 6 comprising an amino acid sequence at least 90% identical to the amino acid sequence of the reference sequence.

9. The polypeptide of claim 5 or 6 comprising an amino acid sequence at least 95% identical to the amino acid sequence of the reference sequence.

10. The polypeptide of any one of claims 5-9, wherein substitutions relative to the reference sequence are selected from the group consisting of SEQ ID NO: 1-8 are selected from those provided in Table 5.

11. The polypeptide of any one of claims 5-9, wherein substitutions relative to the reference sequence are selected from the group consisting of SEQ ID NO:9-23 and 99-100 are selected from those provided in Table 6.

12. The polypeptide of any one of claims 5-9, wherein substitutions relative to the reference sequence are selected from the group consisting of SEQ ID NO:24-32 are selected from those provided in Table 7.

13. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:33-45, wherein the polypeptide binds to TNFR2.

14. The polypeptide of claim 13, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:33-34 and 38-42.

15. The polypeptide of claim 12 or 13 comprising an amino acid sequence at least 75% identical to the amino acid sequence of the reference sequence.

16. The polypeptide of claim 12 or 13 comprising an amino acid sequence at least 90% identical to the amino acid sequence of the reference sequence.

17. The polypeptide of claim 12 or 13 comprising an amino acid sequence at least 95% identical to the amino acid sequence of the reference sequence.

18. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:46 and 47, wherein the polypeptide binds to 0X40.

19. The polypeptide of claim 18 comprising an amino acid sequence at least 75% identical to the amino acid sequence of the reference sequence.

20. The polypeptide of claim 18 comprising an amino acid sequence at least 90% identical to the amino acid sequence of the reference sequence.

21. The polypeptide of claim 18 comprising an amino acid sequence at least 95% identical to the amino acid sequence of the reference sequence.

22. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:48-73 wherein the polypeptide binds to 4-1BB.

23. The polypeptide of claim 13, wherein the polypeptide comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, or 100% the amino acid sequence selected from the group consisting of SEQ ID NO:48-57.

24. The polypeptide of claim 22 or 23 comprising an amino acid sequence at least 75% identical to the amino acid sequence of the reference sequence.

25. The polypeptide of claim 22 or 23 comprising an amino acid sequence at least 90% identical to the amino acid sequence of the reference sequence.

26. The polypeptide of claim 22 or 23 comprising an amino acid sequence at least 95% identical to the amino acid sequence of the reference sequence.

27. The polypeptide of any one of claims 1-26, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or all identified interface residues are identical (not substituted), or conservatively substituted, relative to the reference sequence.

28. The polypeptide of any one of claims 1-27, wherein substitutions relative to the reference sequence are conservative amino acid substitutions.

29. A fusion protein, comprising:(a) the polypeptide of any one of claims 1-28; and(b) one or more functional domains at the N-terminus and / or at the C-terminus of the polypeptide.

30. The fusion protein of claim 29, wherein the one or more functional domains comprising an amino acid sequence at least at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from SEQ ID NO:74-98.

31. The fusion protein of claim 29 or 30, comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOT04-295.

32. The polypeptide or fusion protein of any one of claims 1-31, wherein the polypeptide binds its target with nanomolar or picomolar affinity or better.

33. An oligomer, comprising 2, 3, 4, 5, or more copies of the polypeptide or fusion protein of any one of claims 1-32.

34. The oligomer of claim 33, wherein the polypeptide of any one of claims 1-28 is fused to an oligomerization domain, including but not limited to an oligomerization domain comprising an amino acid sequence at least at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence selected from SEQ ID NO:74-98, wherein the oligomer comprises 2, 3, 4, 5, or more copies of the oligomerization domain-containing fusion protein.

35. A nucleic acid encoding the polypeptide or fusion protein of any one of claims 1-34.

36. An expression vector comprising the nucleic acid of claim 35 operatively linked to a suitable control sequence, such as a promoter.37 A host cell comprising the polypeptide, fusion protein, oligomer, nucleic acid, or expression vector of any one of claims 1-36.

38. A pharmaceutical composition, comprising:(a) the polypeptide, fusion protein, oligomer, nucleic acid, expression vector, and / or host cell of any one of claims 1-37; and(b) a pharmaceutically acceptable carrier.

39. A method for treating an autoimmune disease, including but not limited to rheumatoid arthritis, psoriasis, and inflammatory bowel disease, and / or a neurodegenerative disease where inflammation is a contributing factor (including but not limited to Alzheimer’s disease and multiple sclerosis), comprising administering to a subject in need thereof an amount effective to treat the disorder of the polypeptide of any one claims 5-12, fusion proteins thereof, oligomers thereof, pharmaceutical compositions thereof, or nucleic acids or expression vectors expressing the polypeptide or fusion protein.

40. A method for treating cancer, including but not limited to a melanoma or ovarian cancer, comprising administering to a subject in need thereof an amount effective to treat the cancer of the polypeptide of any one claims 13-17, fusion proteins thereof, pharmaceutical compositions thereof, or nucleic acids or expression vectors expressing the polypeptide or fusion protein.

41. A method for treating an autoimmune disease, including but not limited to rheumatoid arthritis, psoriasis, and inflammatory bowel disease, and / or neurodegenerative disease (including but not limited to Alzheimer’s disease and multiple sclerosis), and / or promoting angiogenesis and cell proliferation in damaged tissues, comprising administeringto a subject in need thereof an amount effective to treat the disorder of an oligomer of the polypeptide of any one claims 13-17, or pharmaceutical compositions thereof.

42. The method of claim 41, further comprising administering the subject the polypeptide of any one claims 5-12, fusion proteins thereof, oligomers thereof, pharmaceutical compositions thereof, or nucleic acids or expression vectors expressing the polypeptide or fusion protein.

43. A method for treating an autoimmune disease, including but not limited to rheumatoid arthritis, psoriasis, and inflammatory bowel disease, and / or graft versus host disease, comprising administering to a subject in need thereof an amount effective to treat the cancer of the polypeptide of any one claims 18-26, fusion proteins thereof, pharmaceutical compositions thereof, or nucleic acids or expression vectors expressing the polypeptide or fusion protein.

44. A method for treating cancer, comprising administering to a subject in need thereof an amount effective to treat the disorder of an oligomer of the polypeptide of any one claims 18-26, or pharmaceutical compositions thereof.

45. An in vitro method for expanding T cells, comprising contacting a T-cell containing culture with an oligomer of the polypeptide of any one claims 18-26, thus expanding the T- cells.

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