Synthetic tnfr1 binding proteins, manufacture, and uses thereof

Abstract

The present disclosure provides synthetic tumor necrosis factor receptor 1 (TNFR1) binding proteins, compositions containing such binding proteins, and methods of making and using such binding proteins, including their uses in treating an inflammatory and / or autoimmune disease.

Description

SYNTHETIC TNFR1 BINDING PROTEINS, MANUFACTURE, AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 505,423, filed May 31, 2023, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes. SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on May 22, 2024, is named AIP-002WO_SL.xml and 1.14 kilobytes in size. FIELD

[0003] The disclosure relates generally to synthetic TNFR1 binding proteins, their manufacture and use in the treatment of various TNFR1-mediated diseases and disorders, including inflammatory and autoimmune disorders. BACKGROUND

[0004] According to the National Institute of Health, autoimmune diseases affect more than 23.5 million people in the United States. Autoimmune diseases include diseases which result from the body’s immune system attacking healthy cells or tissues, and currently include more than one hundred identified types of diseases, including, for example, ankylosing spondylitis, celiac disease, Crohn’s disease, inflammatory bowel disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, ulcerative colitis, and uveitis.

[0005] Current treatment options for autoimmune-mediated diseases often involve generalized immune suppression, achieved through corticosteroids and other immunosuppressive / immunomodulatory medications (e.g., disease modifying antirheumatic drugs) and / or biologics. Combinations of these classes of therapeutics are often used in an attempt to modulate cellular processes and mitigate cellular damage, manage refractory flares, minimize symptoms, and manage expected recurrences in patients.

[0006] Corticosteroids are often used on both a chronic and acute basis to control disease and act through a variety of mechanisms to control or downregulate multiple inflammatory pathways. In many cases, systemic immunosuppressive / immunomodulatory medications such as, e.g., mycophenolate, azathioprine, and methotrexate, are used in combination with IPTS / 128584488.1 Page 1 of 180corticosteroids and / or biologics. However, there can be risk of serious and / or life- threatening infection for subjects receiving chronic immunosuppressive therapy. Furthermore, most therapies for treating autoimmune diseases are not curative and require chronic administration, often for the lifetime of a subject. As such, there is a significant unmet medical need for patients diagnosed with an autoimmune disease.

[0007] Biologics are another established treatment for a variety of autoimmune diseases owing to improved efficacy and safety profiles, as compared to some small molecule therapeutics. Major targets of many biologic therapies for treating autoimmune disorders are cytokines, co-stimulation molecules, and B cells and T cells. Anti-cytokines include, for example, antibodies or antibody fragments against targets such as tumor necrosis factor (TNF) and TNF receptor 1 (TNFR1), interleukin-1 (IL-1), and interleukin-6 (IL-6).

[0008] A number of anti-TNFα antibody and antibody-based therapeutics have been approved in the United States. For example, etanercept (Enbrel®), adalimumab (Humira®), infliximab (Remicade®), and golimumab (Simponi®) have been approved for the treatment of moderate to severe rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. In addition, etanercept and infliximab have also been approved for the treatment of plaque psoriasis, and infliximab and golimumab have also been approved for the treatment of ulcerative colitis, infliximab has also been approved for the treatment of Crohn’s disease, and adalimumab has also been approved for the treatment of hidradenitis suppurativa.

[0009] Other protein-based molecules that interfere with the TNFR1 axis via TNFR1 include, for example, single domain anti-TNFR1 antibodies (dAb) and constructs comprising them (see, e.g., WO 2010 / 094720) and mono scFv derivatives and bivalent TNF receptor antagonists (see, e.g., Brocks et al. (1997) IMMUNOTECH. 3(3):173-184).

[0010] However, certain biologics for treating autoimmune diseases such as those that modulate TNF-pathway components (TNFα and TNFR1), are known to bear risk of pro- inflammatory reactions, including cytotoxicity and apoptosis (see, e.g., Toussirot and Aubin (2016) RMD OPEN 2:e000239; Li, et al. (2017) FRONT. PHARMACOL. 8: 460.). Such pro-inflammatory reactions can be counterproductive when treating an autoimmune disease. As a result, despite the efforts that have been made to date, there remains a need for new and useful treatments for TNFR1-mediated diseases (e.g., autoimmune diseases) including those with lower or different risk profiles compared to currently approved therapies. IPTS / 128584488.1 Page 2 of 180SUMMARY

[0011] The disclosure is based, in part, upon the discovery of synthetic TNFR1 binding proteins that specifically bind TNFR1 and modulate the TNFR1 axis, for example, by preventing ligand binding and reducing or eliminating downstream TNFR1-signaling activity. The TNFR1 binding proteins described herein have specific binding activity for TNFR1, have the ability to reduce downstream TNFR1-mediated signaling activity, and, among other things, are thermally and chemically stable, resistant to oxidation, resistant to protease degradation, deamination, and glycosylation, and have mM-level solubility. The binding proteins can be monovalent, bivalent, or multivalent and can also be conjugated (e.g., via chemical conjugation or as a fusion protein) to an effector molecule.

[0012] Accordingly, the disclosure provides, among other things, synthetic TNFR1 binding proteins, methods of making such binding proteins, and methods of using such proteins to treat a disease or disorder mediated by the TNFR1 axis.

[0013] In one aspect, the disclosure provides a synthetic TNFR1 binding protein, comprising: (a) an amino acid sequence from 30 amino acids to 95 amino acids in length; (b) a net negative charge when present in phosphate buffered saline (PBS); (c) a binding affinity for TNFR1 stronger than 10 ^M; and (d) a stability profile such that the protein (i) retains at least 90% binding affinity to TNFR1 upon cooling to room temperature after thermal denaturation at 95oC in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to TNFR1 after incubation for 16 hours at 37oC of incubation in PBS relative to the protein under the same conditions prior to incubating; and / or (iii) retains at least 90% binding affinity to TNFR1 in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

[0014] In another aspect, the disclosure provides a synthetic TNFR1 binding protein, comprising (a) an amino acid sequence from 30 amino acids to 95 amino acids in length; (b) a net negative charge in PBS; (c) a binding affinity for TNFR1 stronger than 10 ^M; (d) at least three alpha helices; (e) at least two amino acid loops, where a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., a N-terminal amino acid) of a second alpha helix, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., an C-terminal amino acid) of the second alpha helix to a terminal amino acid (e.g., IPTS / 128584488.1 Page 3 of 180an N-terminal amino acid) of a third alpha helix; and (f) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one, two or three of the three alpha helices.

[0015] In certain embodiments, the synthetic TNFR1 binding protein binds to TNFR1 through a paratope of the TNFR1 binding protein, which paratope is defined by amino acids X22 - X26 - X31 - X39, wherein X22 is Q, X26 is selected from L, V, I, or A, X31 is selected from E or D, and X39 is selected from Y or F, and wherein the amino acid numbering corresponds to the numbering of SEQ ID NO: 993 or 1021.

[0016] In one aspect, the disclosure provides a synthetic TNFR1 binding protein comprising a conformational paratope defined by amino acids X22 - X26 - X31 - X39, wherein X22 is Q, X26 is selected from L, V, I, or A, X31 is selected from E or D, and X39 is selected from Y or F, and wherein the amino acid numbering corresponds to the numbering of SEQ ID NO: 993 or 1021.

[0017] In certain embodiments, the synthetic TNFR1 binding protein of claim 3 or 4, wherein X22 is Q, X26 is L, X31 is E, and X39 is Y.

[0018] In some embodiments, the disclosure provides a synthetic TNFR1 binding protein comprising a paratope defined by any amino acid combination set forth in Table 2D or 8.

[0019] In certain embodiments, the synthetic TNFR1 binding protein comprises one or more of the following features: (a) free of tryptophan amino acids; (b) free of methionine amino acids; (c) free of lysine amino acids; (d) does not comprise an unpaired cysteine amino acid when cysteine amino acids are present in the protein; (e) free of glycosylation sites; (f) free of protease cleavage sites; and (g) soluble up to at least 1 mM in PBS at 4oC for one month.

[0020] In certain embodiments, the synthetic TNFR1 binding protein has a binding affinity between about 10 ^M to about 0.1 n ^; about 7.5 ^M to about 0.75 nM; about 5 ^M to about 0.5 nM ^ about 2.5 ^M to about 0.25 nM; about 1 ^M to about 1 n ^; about 0.75 ^M to about 1 nM, about 0.5 ^M to about 1 nM; about 0.25 ^M to about 1 n ^ ^ ^about 0.10 ^M to about 1 n ^ ^ ^about 75 n ^ to about 1 n ^ ^ ^about 50 n ^ to about 1 n ^ ^ ^about 25 n ^ to about 1 n ^ ^ ^about 10 n ^ to about 1 n ^ ^ ^and about 5 n ^ to about 1 n ^. The synthetic TNFR1 binding protein can have a binding affinity stronger than about 10 ^M, about 7.5 ^M, about 5 ^M, about 2.5 ^M, about 1 ^M, about 0.75 ^M, about 0.5 ^M, about 0.25 ^M, about 0.1 IPTS / 128584488.1 Page 4 of 180^M, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, and about 0.1 nM.

[0021] In certain embodiments, the N-terminus of the first alpha helix in the synthetic TNFR1 binding protein is preceded by one or more N-terminal amino acids, and / or the C- terminus of the third alpha helix in the synthetic TNFR1 binding protein is followed by one or more C-terminal amino acids. Depending on the circumstances, the N-terminus of the first alpha helix comprises an N-terminal extension, e.g., comprising 5, 10, 15, 20, 25, 30, 35 or more amino acids in length. For example, in some embodiments, an N-terminal extension comprises an amino acid sequence comprising that of SEQ ID NO: 1026. In certain embodiments, the C-terminus of the third alpha helix is followed by one or more C-terminal amino acids. In some embodiments, the C-terminus of the third alpha helix comprises a C- terminal extension, e.g., comprising 5, 10, 15, 20, 25, 30, 35 or more amino acids in length. For example, in some embodiments, the C-terminal extension comprises an amino acid sequence comprising that of SEQ ID NO: 1029.

[0022] In certain embodiments, the synthetic TNFR1 binding protein comprises from 35 amino acids to 85 amino acids in length, from 35 amino acids to 75 amino acids in length, from 35 amino acids to 65 amino acids in length, from 35 amino acids to 55 amino acids in length, from 35 amino acids to 50 amino acids in length, from 40 amino acids to 85 amino acids in length, from 40 amino acids to 75 amino acids in length, from 40 amino acids to 65 amino acids in length, from 40 amino acids to 55 amino acids in length, or from 40 amino acids to 50 amino acids in length. In certain embodiments, the synthetic TNFR1 binding protein comprises 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0023] In certain embodiments, the synthetic TNFR1 binding protein comprises a first, second, and / or third alpha helix, or a combination thereof, preceded by an alpha helix cap amino acid. In some such embodiments, each alpha helix cap amino acid is independently selected from the group consisting of serine, threonine, aspartate, and asparagine.

[0024] In certain embodiments, the synthetic TNFR1 binding protein comprises one or more of the following: (a) the first, second, and / or third alpha helix each contains at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids (e.g., a hydrophobic amino acid that is not solvent accessible); (b) the first, second, and / or third alpha helix each contains at least two or three hydrophobic amino acids, IPTS / 128584488.1 Page 5 of 180wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible; (c) the first, second, and / or third alpha helix each contain at least one or two solvent accessible amino acids; (d) the first and / or second loop contains at least one hydrophobic amino acid, or (e) the binding protein comprises any combination of elements selected from (a), (b), (c), and (d). In some such embodiments, (a) the second and third alpha helix each contains at least two hydrophobic amino acids; (b) the first, second, and third alpha helix each contains at least one solvent accessible amino acid; (c) the first, second, and third alpha helix each contain at least two hydrophobic and one solvent accessible amino acids; (e) the second and third alpha helix each contains at least four solvent accessible amino acids; and / or (f) the first loop contains at least one hydrophobic amino acid.

[0025] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively; and (ii) an amino acid of SEQ ID NO: 1, wherein D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of SEQ ID NO: 3, wherein X4 is E or D; X6 is L or I; X7 is E, R, or Q; X8 is Q, R, or E; and X11 is D or E; (b) D2 comprises an amino acid sequence of SEQ ID NO: 4, wherein X20 is Y or A; X21 is Q, R, E, or D; X24 is L, E, I, V, or T; and X28 is D or E; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 5, wherein X33 is P or E; X34 is Q or E; X37 is R or I; X41 is E, D, N, or R; X43 is Y or H; and X45 is R or E.

[0026] In certain embodiments, the synthetic TNFR1 binding protein comprises L1, which comprises an amino acid sequence of X12GX14IS and L2, which comprises an amino acid sequence of X29GED, wherein X12 is Q, E, R, or N; X14 is L, R, or E; and X29 is R, E, or Q.

[0027] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising an amino acid sequence of SEQ ID NO: 1, wherein X4 is E or D; X6 is L or I; X7 is E, R, or Q; X8 is Q, R, or E; X11 is D or E; X12 is Q, E, R, or N; X14 is L, R, or E; X20 is Y, or A; X21 is Q, R, E, or D; X24 is L, E, I, V, or T; X28 is D or E; X29 is R, E, or Q; X33 is P or E; X34 is Q or E; X37 is R or I; X41 is E, D, N, or R; X43 is Y or H; and X45 is R or E.

[0028] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising: an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 IPTS / 128584488.1 Page 6 of 180(Formula I), wherein, D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively, and wherein D1, D2, and D3 independently comprise any of the following combinations: D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11, wherein X2 is A or G; X3 is R, A, D, Q, Y, K, H, S, L, N, T, G, or V; X4 is D, A, E, K, S, I, N, V, T, L, Q, F, R, H, Y, or G; X5 is Y, T, G, I, Q, R, A, H, V, S, E, F, K, or D; X7 is E, L, A, R, Q, I, D, K, F, G, V, T, H, N, S, or Y; X8 is R, Y, E, L, V, A, F, Q, N, K, H, T, or G; X10 is R, A, T, V, E, or Y; and X11 is R, D, E, K, S, L, Q, T, F, I, V, G, H, or N; D2 comprises an amino acid sequence of X17X18LX20X21QLX24X25X26X27X28, wherein X17 is D, F, Y, G, H, T, R, A, K, S, H, N, E, V, Q, or I; X18 is V, I, T, Y, F, or D; X20 is E, Y, Q, H, K, N, X, R, V, T, A, S, F, or L; X21 is Q, Y, D, G, H, I, E, T, V, N, F, R, S, or A; X24 is I, S, N, V, G, T, Q, E, D, L, R, F, Y, or A; X25 is D, S, Y, E, Q, or T; X26 is L, I, or V; X27 is L, I, or V; and X28 is N, L, D, R, T, I, Y, A, E, Q, G, V, F, or H; and D3 comprises an amino acid sequence set forth in SEQ ID NO: 21, wherein X33 is E, G, S, A, D, Y, F, Q, I, R, H, K, or N; X34 is N, Q, H, R, Y, V, I, D, A, L, F, K, S, E, or G; X37 is I, V, T, E, R, A, K, or S; X41 is N, Y, L, I, H, D, F, Q, V, S, R, E, A, or T; X43 is Y or F; X44 is I or L; and X45 is E, D, T, R, F, L, S, A, I, Y, V, or N.

[0029] In certain embodiments, the synthetic TNFR1 binding comprises L1, which comprises an amino acid sequence of X12X13X14IX16; and L2, which comprises an amino acid sequence of X29GEX32, wherein X12 is E, Q, S, F, I, K, T, R, L, Y, A, D, N, G, V, or H; X13 is G, R, H, K, S, L, Q, N, I, or Y; X14 is R, Y, G, A, L, I, H, F, V, K, T, E, Q, S, or N; X16 is S, N, G, D, T, or E; X29 is R, I, Q, N, E, S, K, V, L; and X32 is D, F, Y, Q, E, R, N, L, H, A, I, S, V, G, T, or K.

[0030] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising: an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein, D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively, and D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of AX3X4X5LX7X8X9RX11, wherein X3 is R, A, D, Q, Y, or K; X4 is D, A, E, K, S, or I; X5 is Y, T, or G; X7 is E, L A, R, Q, I, D, K, or F; X8 is R or Y; X9 is L or Y; and X11 is R, D, E, K, S or L; D2 comprises an IPTS / 128584488.1 Page 7 of 180amino acid sequence of SEQ ID NO: 24, wherein X17 is D, F, Y, G, or H; X20 is E, Y, Q, H, or K; X21 is Q, Y, D, G, or H; X24 is I, S, N, V, or G; X25 is D, S, or Y; X27 is L or I; and X28 is N, L, D or R; and D3 comprises an amino acid sequence of SEQ ID NO: 25, wherein X33 is E, G, or S; X34 is N, Q, or H; X41 is N, Y, L, or I; X43 is Y or F; and X45 is E, D, T or R; (b) D1 comprises an amino acid sequence of SEQ ID NO: 50, wherein X4 is D or E; and X7 is E or K; D2 comprises an amino acid sequence of SEQ ID NO: 51, wherein X17 is Y, G, or D; and X20 is K, Q, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 52, wherein X33 is E or S; X34 is Q or R; and X41 is N, H, or D; (c) D1 comprises an amino acid sequence of SEQ ID NO: 75, wherein X3 is R, H, S, or K; X4 is D, N, V, T, S, E, L, Q, F, K, or I; X5 is I, Y, Q, or R; X7 is E, L, G, A, K, F, V, Q, or R; X8 is R, Y, E, L, V, or A; and X11 is D, K, Q, or T; D2 comprises an amino acid sequence of SEQ ID NO: 76, wherein X17 is D, G, Y, or H; X18 is V, I, or T; X20 is Y, K, R, V, T, E, Q, or A; X21 is Q, G, D, or H; X24 is I, T, or G; X25 is D, E, or Q; and X28 is D, L, T, I, Y, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 77, wherein X33 is A, S, D, E, Y, or F; X34 is Q, Y, V, I, or D; X41 is Y, N, or D; X43 is Y or F; and X45 is E, D, F, or L; (d) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11, wherein X2 is A or G; X3 is R, L, or A; X4 is D, E, K, or R; X5 is A or Y; X7 is E, G, R, L, K, or V; X8 is R, F, Y, or Q; X10 is R or A; and X11 is D or Q; D2 comprises an amino acid sequence of SEQ ID NO: 150, wherein X17 is F, H, T, D, R, A, K, S, or Y; X21 is Q or I; X24 is Q, I, E, or V; X28 is D or N; and D3 comprises an amino acid sequence of SEQ ID NO: 151, wherein X33 is E, G, Y, Q, I, A, F, S, or R; X37 is I or V; X41 is N, F, Y, or L; and X43 is Y or F; (e) D1 comprises an amino acid sequence of SEQ ID NO: 194, wherein X3 is R, D, A, H, N, Q or T; X4 is D, R, N, E, or Q; X7 is E, K, Q, G, R, L, or I; X8 is R, E, A, N, or Q; and X11 is D, F, I, or K; D2 comprises an amino acid sequence of SEQ ID NO: 195, wherein X17 is S, F, Y, H, A, G, R, N, E, D, or T; X18 is V, Y, F, D, or T; X20 is Y, Q, or R; X21 is Q, Y, E, H, or T; X24 is S, I, V, E, or N; X25 is D or E; X26 is L or V; X27 is L or I; and X28 is D, A, or L; and D3 comprises an amino acid sequence of SEQ ID NO: 196, wherein X33 is E, D, F, G, or Y; X34 is Q, I, A, V, N, D, or L; X37 is I or T; X43 is Y or F; and X45 is E, F, or S; IPTS / 128584488.1 Page 8 of 180(f) D1 comprises an amino acid sequence of AX3X4X5LX7X8LX10X11, wherein X3 is R, Y, A, S, Q, or G; X4 is Q, E, F, D, K, R, or Y; X5 is Y, T, or A; X7 is E, R, G, T, L, H, F, K, A, I, or V; X8 is R, K, or Y; X10 is R or T; and X11 is D or V; D2 comprises an amino acid sequence of SEQ ID NO: 274, wherein X17 is F, D, Y, H, K, S, R, V, A, G, or I; X18 is V or T; X20 is Y, R, E, V, K, H, or T; X21 is Q, Y, V, H, G, or N; X24 is D, I, G, N, or L; X25 is D or E; X26 is L, I, or V; X27 is L or V; and X28 is E, L, D, Q, T, G, or I; and D3 comprises an amino acid sequence of SEQ ID NO: 275, wherein X33 is E, S, D, G, A, H, Y, K, or N; X34 is Q, Y, F, N, R, H, I, K, or Z; X37 is I, E, R, or A; X41 is Y, F, N, L, or I; X43 is Y or F; X44 is I or L; and X45 is E or A; (g) D1 comprises an amino acid sequence of SEQ ID NO: 346, wherein X4 is D, F, or H; and X7 is K, E, G, or R; D2 comprises an amino acid sequence of SEQ ID NO: 347, wherein X17 is Y, H, V, or F; X20 is E, K, or Y; X24 is I or S; X26 is L or V; and X27 is L or V; and D3 comprises an amino acid sequence of SEQ ID NO: 348, wherein X34 is F, N, Y, or H; X37 is I or R; X41 is Y or N; and X45 is E, D, or A; (h) D1 comprises an amino acid sequence of SEQ ID NO: 368, wherein X3 is R or H; X4 is D, Y, N, I, or H; X5 is Y or H; X7 is E, A, or K; X8 is R, A, or H; X11 is D, K, or R; D2 comprises an amino acid sequence of SEQ ID NO: 369, wherein X17 is A, G, Q, D, Y, T, N, F, R, K, H, or I; and D3 comprises an amino acid sequence of SEQ ID NO: 370, wherein X34 is H, N, Y, E, G, D, R, F, A, or I; and X41 is L, D, N, Y, or F; (i) D1 comprises an amino acid sequence of SEQ ID NO: 405, wherein X3 is R or Q; X4 is D, Q, V, or S; X5 is Y, T, or V; X7 is E, R, or G; X8 is R or N; and X11 is D, F, or G; D2 comprises an amino acid sequence of SEQ ID NO: 406, wherein X17 is G, Y, A, T, R, F, S, I, Q, or H; X18 is Y or V; X20 is Y, K, E, or T; and X21 is Q or F; and D3 comprises an amino acid sequence of SEQ ID NO: 407, wherein X34 is F, I, D, S, H, R, N, or Y; and X41 is Q, V, Y, N, S, L, I, F, D, R, E, or H; (j) D1 comprises an amino acid sequence of AX3X4X5LX7X8LX10X11, wherein X3 is R, Q, K, A, or E; X4 is D, H, G, S, K, I, L, E, T, or V; X5 is Y, S, E, V, or F; X7 is E, R, G, I, L, or K; X8 is R, T, or G; X10 is R or V; and X11 is D, F, or H; D2 comprises an amino acid sequence of SEQ ID NO: 447, wherein X17 is H, Y, S, G, R, K, A, T, F, or N; X18 is V, D, or I; X20 is Y or Q; X21 is G, Y, Q, H, or F; X24 is I, G, L, V, S, R, or D; X25 is Q, D, or E; X26 is L, I, or V; X27 is L, I, or V; and X28 is D, IPTS / 128584488.1 Page 9 of 180A, L, R, E, T, V, F, G, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 448, wherein X33 is E, H, N, S, A, or G; X34 is Q, E, R, or V; X37 is I, A, E, or V; X41 is V, Q, F, Y, N, I, L, D, or E; X43 is Y or F; and X45 is E, A, S, L, I, F, or Y; (k) D1 comprises an amino acid sequence of SEQ ID NO: 524, wherein X4 is D, I, N, H, L, K, or A; X5 is K, E, or Y; X8 is R, H, or V; and X11 is D or I; D2 comprises an amino acid sequence of SEQ ID NO: 525, wherein X17 is R, F, N, K, Y, G, H, S, T, or A; X20 is Y, E, N, Q, K, S, or H; X21 is Q, N, E, G, or R; X24 is I or G; and X26 is L or V; and D3 comprises an amino acid sequence of SEQ ID NO: 526, wherein X34 is Q or F; and X41 is Y, F, I, L, D, V, A, E, S, N, or T; (l) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LRX11, wherein X2 is A or G; X3 is R or Q; X4 is D, S, F, L, T, K, or A; X5 is Y or F; X7 is E, G, T, V, or R; X8 is R or L; and X11 is D or V; D2 comprises an amino acid sequence of SEQ ID NO: 565, wherein X17 is D or Q; X18 is V or I; X20 is Y, A, E, Q, T, or R; X21 is Q or G; X24 is I, F, G, or Y; and X28 is D, H, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 566, wherein X33 is E, S, G, Y, or D; and X34 is Q or E; X37 is I, A, or V; X41 is F, D, V, Y, I, or L; X43 is Y or F; and X45 is E, Y, I, V, or F; (m) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11, wherein X2 is A or G; X3 is R, Y, S, or A; X4 is E, D, Y, T, A, L, G, F, K, N, or R; X5 is Y, A, I, V, E, G, or S; X7 is E, Q, R, N, A, S, K, or T; X8 is R, G, F, L, Q, K, A, or N; X10 is T, R, V, or E; and X11 is D, L, H, Q, I, N, or F; D2 comprises an amino acid sequence of SEQ ID NO: 605, wherein X17 is H, Q, D, Y, F, T, G, or K; X20 is Y, H, S, K, R, A, E, V, Q, F, L, or T; X21 is Q, G, H, or E; X24 is I, A, or R; X25 is D, T, or Q; and X28 is D, R, I, or F; and D3 comprises an amino acid sequence of SEQ ID NO: 606, wherein X33 is E, S, or G; X34 is L, R, Q, S, or H; X37 is I, A, K, S, or V; X41 is Y, I, L, F, V, D, A, H, or T; X43 is Y or F; X44 is I or L; X45 is E, I, N, Y, or L; (n) D1 comprises an amino acid sequence of SEQ ID NO: 677, wherein X3 is R, Q, V, or G; X4 is D, G, F, R, V, Q, E, L, Y, A, or T; X5 is Y, A, I, V, G, E, or H; X7 is K, E, Y, Q, A, S, L; X8 is R, F, Q, T, K, or Y; and X11 is D, L, R, F, N, Q, K, or V; D2 comprises an amino acid sequence of SEQ ID NO: 678, wherein X17 is F, Q, D, G, or Y; X18 is V, D, or T; X20 is Y, H, Q, R, K, A, T, V, L, or E; and X21 is Q, G, H, or Y; and D3 comprises an amino acid sequence of SEQ ID NO: 679, wherein X34 is K, Y, L, H, F, N, A, R, G, D, E, or I; X37 is I or A; and X41 is V, N, I, F, Y, L, E, or H; IPTS / 128584488.1 Page 10 of 180(o) D1 comprises an amino acid sequence of SEQ ID NO: 739, wherein X3 is R, V, or H; X4 is D, E, H, G, or K; X5 is Y, D, G, V, or S; X7 is E or R; and X8 is R, F, or V; D2 comprises an amino acid sequence of SEQ ID NO: 740, wherein X18 is V or I; X20 is T, Y, F, K, or V; X21 is Q, R, S, or H; and X25 is Q or D; and D3 comprises an amino acid sequence of SEQ ID NO: 741, wherein X34 is Y, S, G, R, D, F, N, V, A, L, H, I, or K; and X41 is N, F, L, I, Y, V, or D; (p) D1 comprises an amino acid sequence of SEQ ID NO: 787, wherein X4 is D or I; X5 is Y or T; and X8 is R or K; D2 comprises an amino acid sequence of SEQ ID NO: 788, wherein X1 is G, H, D, or Y; X20 is K or Y; and X21 is Q or Y; and D3 comprises an amino acid sequence of SEQ ID NO: 789, wherein X34 is Q, Y, L, N, or G; and X41 is N, F, I, Y, L, V, or D; (q) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11, wherein X2 is A or G; X3 is R, H, or Y; X4 is D, Y, F, L, or K; X5 is Y, I, S, A, L, T, or V; X7 is E, Y, I, L, K, S, R, G, A, or Q; X8 is R, L, T, Y, V, or E; X10 is R or Y; and X11 is D, H, or R; D2 comprises an amino acid sequence of SEQ ID NO: 813, wherein X18 is T or V; X20 is Y, Q, K, R, T, S, N, A, or F; and X21 is Q, A, G, H, E, or S; and D3 comprises an amino acid sequence of SEQ ID NO: 814, wherein X41 is V, F, D, Y, I, S, E, or L; (r) D1 comprises an amino acid sequence of SEQ ID NO: 875, wherein X4 is G or D; and X7 is E or A; D2 comprises an amino acid sequence of SEQ ID NO: 876, wherein X17 is H, Y, or G; and X20 is Y, K, or R; and D3 comprises an amino acid sequence of SEQ ID NO: 877, wherein X34 is Q, N, or E; and X41 is N, L, Y, F, D, or I; and (s) D1 comprises an amino acid sequence of SEQ ID NO: 897, wherein X2 is A or G; X3 is R, Y, K, or H; X4 is D, S, L, or G; X5 is Y, T, or K; X7 is R, K, I, E, G, or A; and X11 is D, T, or S; D2 comprises an amino acid sequence of SEQ ID NO: 898, wherein X17 is D, H, Y, F, A, Q, T, R, G, I, E, V, or S; X18 is V, I, or F; X20 is Y, K, T, or S; or X21 is Q, A, Y, or G; and D3 comprises an amino acid sequence of SEQ ID NO: 899, wherein X41 is N, L, D, Y, T, V, K, I, A, E, or F.

[0031] Using the similar labeling of miniproteins (a) through (s) above, the miniproteins optionally further comprise linker sequences (denoted as L1 and L2) as follows: (a) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E, Q, S, or F; X13 is G or R; X14 IPTS / 128584488.1 Page 11 of 180is R, Y, G, A, or L; X16 is S, N, G, or D; X29 is R, I, Q, N, E, S, or K; and X32 is D, F, or Y; (b) L1 comprises an amino acid sequence of SEQ ID NO: 990; and L2 comprises an amino acid sequence of X29GEX32, wherein X29 is R, E, or S; and X32 is Q, E, Y, or R; (c) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, I, K, T, F, R, S, L, Q, Y, or A; X13 is G, H, K, S, or L; X14 is R, L, I, H, Y, F, A, V, or K; X16 is S, D, or T; and X32 is R, F, Q, Y, N, D, L, H, or A; (d) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, D, I, T, R, N, Y, or K; X13 is G or R; X14 is R or K; and X32 is Y, I, R, or L; (e) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E, N, F, G, S, Y, or V; X13 is G, H, R, K, or Q; X14 is F, R, K, H, I, T, Y, E, or Q; X29 is R, K, or N; and X32 is L, A, R, N, Q, S, Y, F, or V; (f) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of X29GED, wherein X12 is E, F, R, G, Y, L, or H; X13 is G, K, N, H, or R; X14 is R, F, H, Q, or Y; and X29 is R, V, K, or L; (g) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, N, F, D, I, or K; X13 is G, R, or K; X14 is R, E, F, or K; and X32 is D or A; (h) L1 comprises an amino acid sequence of SEQ ID NO: 991; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X14 is R, A, Y, K, or L; (i) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, T, S, or D; X13 is G, R, or K; and X14 is R, L, or S; (j) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E, T, H, V, K, N, R, L, or F; X13 is G, N, R, H, or K; X14 is R, V, E, Q, H, K, Y, or F; and X32 is D, Q, or R; IPTS / 128584488.1 Page 12 of 180(k) L1 comprises an amino acid sequence of X12X13RIS; and L2 comprises an amino acid sequence of X29GED, wherein X12 is E, G, S, or K; X13 is G or R; and X29 is R or L; (l) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, L, Q, G, D, Y; X13 is G, H, or I; X14 is Y, F, L, H, V, or I; (m) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, K, T, R, I, G, Y, or V; X13 is G, Q, R, K, or H; X14 is R, Y, or K; and X16 is S or E; (n) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, D, F, T, R, S, N, Y, G, L, Q, or K; X13 is G, K, or Y; X14 is H or R; and X16 is S, T, or D; (o) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, Y, S, G, N, I, L; X13 is G, K, or N; X14 is F, Y, A, G, Q, L, I, V, H, K; and X16 is S or D; (p) L1 comprises an amino acid sequence of EX13X14IS; and L2 comprises an amino acid sequence of RGEX32, wherein X13 is G, H, or R; X14 is R or Y; and X32 is D, G, S, F, or Y; (q) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, L, A, Q, I, H, F, or Y; X13 is G, R, H; X14 is Q, R, F, Y, H, or S; X16 is S, N, or D; and X32 is Y, N, V, F, R, L, S, T, Q, or I; (r) L1 comprises an amino acid sequence of EX13X14IS; and L2 comprises an amino acid sequence of RGEX32, wherein X13 is G or K; X14 is R, H, F, or Y; X32 is Y, R, F, or D; and (s) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, K, S, or V; X13 is H, G, or R; X14 is R, H, I, Q, V, Y, F, N, or A; X16 is S or D; and X32 is F, N, Y, A, S, L, Q, R, I, E, K.

[0032] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising an amino acid sequence of SEQ ID NO: 18, wherein X1 is A, S, D, E, F, G, H, I, IPTS / 128584488.1 Page 13 of 180K, L, N, Q, R, S, V, or Y; X2 is A or G; X3 is R, A, D, Q, Y, K, H, S, L, N, T, G, or V; X4 is D, A, E, K, S, I, N, V, T, L, Q, F, R, H, Y, or G; X5 is Y, T, G, I, Q, R, A, H, V, S, E, F, K, or D; X7 is E, L, A, R, Q, I, D, K, F, G, V, T, H, N, S, or Y; X8 is R, Y, E, L, V, A, F, Q, N, K, H, T, or G; X10 is R, A, T, V, E, or Y; X11 is R, D, E, K, S, L, Q, T, F, I, V, G, H, or N; X12 is E, Q, S, F, I, K, T, R, L, Y, A, D, N, G, V, or H; X13 is G, R, H, K, S, L, Q, N, I, or Y; X14 is R, Y, G, A, L, I, H, F, V, K, T, E, Q, S, or N; X16 is S, N, G, D, T, or E; X17 is D, F, Y, G, H, T, R, A, K, S, H, N, E, V, Q, or I; X18 is V, I, T, Y, F, or D; X20 is E, Y, Q, H, K, N, X, R, V, T, A, S, F, or L; X21 is Q, Y, D, G, H, I, E, T, V, N, F, R, S, or A; X24 is I, S, N, V, G, T, Q, E, D, L, R, F, Y, or A; X25 is D, S, Y, E, Q, or T; X26 is L, I, or V; X27 is L, I, or V; X28 is N, L, D, R, T, I, Y, A, E, Q, G, V, F, or H; X29 is R, I, Q, N, E, S, K, V, or L; X32 is D, F, Y, Q, E, R, N, L, H, A, I, S, V, G, T, or K; X33 is E, G, S, A, D, Y, F, Q, I, R, H, K, or N; X34 is N, Q, H, R, Y, V, I, D, A, L, F, K, S, E, or G; X37 is I, V, T, E, R, A, K, or S; X41 is N, Y, L, I, H, D, F, Q, V, S, R, E, A, or T; X43 is Y or F; X44 is I or L; X45 is E, D, T, R, F, L, S, A, I, Y, V, or N; and X46 is S, G, R, D, T, E, L, I, F, V, or A; X47 is R, V, I, L, T, Y, E, S, K, G, Q, D, N, A, or H.

[0033] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising: (a) an amino acid sequence of SEQ ID NO: 22, wherein X1 is F, S, R, D, or L; X3 is R, A, D, Q, Y, or K; X4 is D, A, E, K, S, or I; X5 is Y, T, or G; X7 is E, L, A, R, Q, I, D, K, or F; X8 is R or Y; X9 is L or Y; X11 is R, D, E, K, S, or L; X12 is E, Q, S, or F; X13 is G or R; X14 is R, Y, G, A, or L; X16 is S, N, G, or D; X17 is D, F, Y, G, or H; X20 is E, Y, Q, H, or K; X21 is Q, Y, D, G, or H; X24 is I, S, N, V, or G; X25 is D, S, or Y; X27 is L, or I; X28 is N, L, D, or R; X29 Is R, I, Q, N, E, S, or K; X32 is D, F, or Y; X33 is E, G, or S; X34 is N, Q, or H; X41 is N, Y, L, or I; X43 is Y or F; X45 is E, D, T, or R; X46 is S, G, or R; and X47 is R, V, I, L, T, Y, E, or S; (b) an amino acid sequence of SEQ ID NO: 49, wherein X1 is S, F, V, or R; X4 is D or E; X7 is E or K; X17 is Y, G, D; X20 is K, Q, or N; X29 is R, E, or S; X32 is Q, E, Y, or R; X33 is E or S; X34 is Q or R; X41 is N, H, or D; X46 is S, D, or R; and X47 is K, R, E, L, G, Q, or I; (c) an amino acid sequence of SEQ ID NO: 74, wherein X1 is L, S, Y, F, G, T, I, V, K, H, D, Q, or R; X3 is R, H, S, or K; X4 is D, N, V, T, S, E, L, Q, F, K, or I; X5 is I, Y, Q, or R; X7 is E, L, G, A, K, F, V, Q, or R; X8 is R, Y, E, L, V, or A; X11 is D, K, Q, IPTS / 128584488.1 Page 14 of 180or T; X12 is E, I, K, T, F, R, S, L, Q, Y, or A; X13 is G, H, K, S, or L; X14 is R, L, I, H, Y, F, A, V, or K; X16 is S, D, or T; X17 is D, G, Y, H; X18 is V, I, or T; X20 is Y, K, R, V, T, E, Q, or A; X21 is Q, G, D, or H; X24 is I, T, or G; X25 is D, E, or Q; X28 is D, L, T, I, Y, or N; X32 is R, F, Q, Y, N, D, L, H, or A; X33 is A, S, D, E, Y, or F; X34 is Q, Y, V, I, D; X41 is Y, N, or D; X43 is Y or F; X45 is E, D, F, or L; X46 is S, D, T, E, or L; and X47 is R, T, L, Q, I, D, S, Y, or N; (d) an amino acid sequence of SEQ ID NO: 148, wherein X1 is S or V; X2 is A or G; X3 is R, L, or A; X4 D, E, K, or R; X5 is A or Y; X7 is E, G, R, L, K, or V; X8 is R, F, Y, or Q; X10 is R or A; X11 is D or Q; X12 is E, D, I, T, R, N, Y, or K; X13 is G or R; X14 is R or K; X17 is F, H, T, D, R, A, K, S, or Y; X21 is Q or I; X24 is Q, I, E, or V; X28 is D or N; X32 is Y, I, R, or L; X33 is E, G, Y, Q, I, A, F, S, or R; X37 is I or V; X41 is N, F, Y, or L; X43 is Y or F; X46 is S or G; X47 is R, T, E, Q, L, N, Y, I, or F; (e) an amino acid sequence of SEQ ID NO: 193, wherein X1 is S, V, Y, F, K, R, G, or A; X3 is R, D, A, H, N, Q, or T; X4 is D, R, N, E, or Q; X7 is E, K, Q, G, R, L, or I; X8 is R, E, A, N, or Q; X11 is D, F, I, or K; X12 is E, N, F, G, S, Y, or V; X13 is G, H, R, K, or Q; X14 is F, R, K, H, I, T, Y, E, or Q; X17 is S, F, Y, H, A, G, R, N, E, D, or T; X18 is V, Y, F, D, or T; X20 is Y, Q, or R; X21 is Q, Y, E, H, or T; X24 is S, I, V, E, or N; X25 is D or E; X26 is L or V; X27 is L or I; X28 is D, A, or L; X29 is R, K, or N; X32 is L, A, R, N, Q, S, Y, F, or V; X33 is E, D, F, G, or Y; X34 Q, I, A, V, N, D, or L; X37 is I or T; X43 is Y or F; X45 is E, F, or S; X46 is S, I, E, F, V, A, or D; and X47 Is R, A, E, T, L, I, N, G, Q, or S; (f) an amino acid sequence of SEQ ID NO: 272, wherein X1 is S, R, K, F, or L; X3 is R, Y, A, S, Q, or G; X4 is Q, E, F, D, K, R, or Y; X5 is Y, T, or A; X7 is E, R, G, T, L, H, F, K, A, I, or V; X8 is R, K, or Y; X10 is R or T; X11 is D or V; X12 is E, F, R, G, Y, L, or H; X13 is G, K, N, H, or R; X14 is R, F, H, Q, or Y; X16 is S or D; X17 F, D, Y, H, K, S, R, V, A, G, or I; X18 is V or T; X20 is Y, R, E, V, K, H, or T; X21 is Q, Y, V, H, G, or N; X24 is D, I, G, N, or L; X25 is D or E; X26 is L, I, or V; X27 is L or V; X28 is E, L, D, Q, T, G, or I; X29 is R, V, K, or L; X33 is E, S, D, G, A, H, Y, K, or N; X34 is Q, Y, F, N, R, H, I, K, or A; X37 is I, E, R, or A; X41 is Y, F, N, L, or I; X43 is Y or F; X44 is I or L; X45 is E or A; X46 is S, L, D, I, E, or A; X47 is R, V, L, T, I, G, Q, E, F, S, or Y; IPTS / 128584488.1 Page 15 of 180(g) an amino acid sequence of SEQ ID NO: 345, wherein X4 is D, F, or H; X7 is K, E, G, or R; X12 is E, N, F, D, I, or K; X13 is G, R, or K; X14 is R, E, F, or K; X17 is Y, H, V, or F; X20 is E, K, or Y; X24 is I or S; X26 is L or V; X27 is L or V; X32 is D or A; X34 is F, N, Y, or H; X37 is I or R; X41 is Y or N; X45 is E, D, or A; X46 is S, L, or F; X47 is R, L, V, Q, E, N, or I; (h) an amino acid sequence of SEQ ID NO: 367, wherein X1 is S, F, or V; X3 is R or H; X4 is D, Y, N, I, or H; X5 is Y or H; X7 is E, A, or K; X8 is R, A, or H; X11 is D, K, or R; X14 is R, A, Y, K, or L; X17 is A, G, Q, D, Y, T, N, F, R, K, H, or I; X18 is V, D, F, or T; X20 is H, K, V, or Y; X21 is Q, H, or Y; X34 is H, N, Y, E, G, D, R, F, A, or I; X41 is L, D, N, Y, or F; and X47 is R or I; (i) an amino acid sequence of SEQ ID NO: 404, wherein X1 is S, I, or Y; X3 is R or Q; X4 is D, Q, V, is S; X5 is Y, T, or V; X7 is E, R, or G; X8 is R or N; X11 is D, F, or G; X12 is E, T, S, or D; X13 is G, R, or K; X14 is R, L, or S; X17 is G, Y, A, T, R, F, S, I, Q, or H; X18 is Y or V; X20 is Y, K, E, or T; X21 is Q or F; X34 is F, I, D, S, H, R, N, or Y; X41 is Q, V, Y, N, S, L, I, F, D, R, E, or H; (j) an amino acid sequence of SEQ ID NO: 445, wherein X1 is S, Q, F, R, or K; X3 is R, Q, K, A, or E; X4 is D, H, G, S, K, I, L, E, T, V; X5 is Y, S, E, V, or F; X7 is E, R, G, I, L, or K; X8 is R, T, or G; X10 is R or V; X11 is D, F, or H; X12 is E, T, H, V, K, N, R, L, or F; X13 is G, N, R, H, or K; X14 is R, V, E, Q, H, K, Y, or F; X17 is H, Y, S, G, R, K, A, T, F, or N; X18 is V, D, or I; X20 is Y or Q; X21 is G, Y, Q, H, or F; X24 is I, G, L, V, S, R, or D; X25 is Q, D, or E; X26 is L, I, or V; X27 is L, I, or V; X28 is D, A, L, R, E, T, V, F, G, or N; X29 is R or K; X32 is D, Q, or R; X33 is E, H, N, S, A, or G; X34 is Q, E, R, or V; X37 is I, A, E, or V; X41 is V, Q, F, Y, N, I, L, D, or E; X43 is Y or F; X45 is E, A, S, L, I, F, or Y; X46 is S, L, or E; and X47 is R or Y; (k) an amino acid sequence of SEQ ID NO: 523, wherein X1 is S or K; X4 is D, I, N, H, L, K, or A; X5 is K, E, or Y; X8 is R, H, or V; X11 is D or I; X12 is E, G, S, or K; X13 is G or R; X17 is R, F, N, K, Y, G, H, S, T, or A; X20 is Y, E, N, Q, K, S, or H; X21 is Q, N, E, G, or R; X24 is I or G; X26 is L or V; X29 is R or L; X34 is Q or F; and X41 is Y, F, I, L, D, V, A, E, S, N, or T; (l) an amino acid sequence of SEQ ID NO: 563, wherein X1 is V, S, or L; X2 is A or G; X3 is R or Q; X4 is D, S, F, L, T, K, or A; X5 is Y or F; X7 is E, G, T, V, or R; X8 is R or L; X11 is D or V; X12 is E, L, Q, G, D, or Y; X13 is G, H, or I; X14 is Y, F, L, IPTS / 128584488.1 Page 16 of 180H, V, or I; X17 is D or Q; X18 is V or I; X20 is Y, A, E, Q, T, or R; X21 is Q or G; X24 is I, F, G, or Y; X28 is D, H, or N; X33 is E, S, G, Y, or D; X34 is Q or E; X37 is I, A, or V; X41 is F, D, V, Y, I, or L; X43 is Y or F; X45 is E, Y, I, V, or F; X46 is S, D, E, L, or A; and X47 is S, V, R, E, or L; (m) an amino acid sequence of SEQ ID NO: 603, wherein X1 is S, R, K, I, Q, L, Y, or F; X2 is A or G; X3 is R, Y, S, or A; X4 is E, D, Y, T, A, L, G, F, K, N, or R; X5 is Y, A, I, V, E, G, or S; X7 is E, Q, R, N, A, S, K, or T; X8 is R, G, F, L, Q, K, A, or N; X10 is T, R, V, or E; X11 is D, L, H, Q, I, N, or F; X12 is E, K, T, R, I, G, Y, or V; X13 is G, Q, R, K, or H; X14 is R, Y, or K; X16 is S or E; X17 is H, Q, D, Y, F, T, G, or K; X20 is Y, H, S, K, R, A, E, V, Q, F, L, or T; X21 is Q, G, H, or E; X24 is I, A, or R; X25 is D, T, or Q; X28 is D, R, I, or F; X33 is E, S, or G; X34 is L, R, Q, S, or H; X37 is I, A, K, S, or V; X41 is Y, I, L, F, V, D, A, H, or T; X43 is Y or F; X44 is I or L; X45 is E, I, N, Y, or L; X46 is S, E, D, or L; and X47 is R, H, E, I, A, V, L, S, T, Q, or K; (n) an amino acid sequence of SEQ ID NO: 676, wherein X1 is S, G, K, V, Q, or E; X3 is R, Q, V, or G; X4 is D, G, F, R, V, Q, E, L, Y, A, or T; X5 is Y, A, I, V, G, E, or H; X7 is K, E, Y, Q, A, S, or L; X8 is R, F, Q, T, K, or Y; X11 is D, L, R, F, N, Q, K, or V; X12 is E, D, F, T, R, S, N, Y, G, L, Q, or K; X13 is G, K, or Y; X14 is H or R; X16 is S, T, or D; X17 is F, Q, D, G, or Y; X18 is V, D, or T; X20 is Y, H, Q, R, K, A, T, V, L, or E; X21 is Q, G, H, or Y; X34 is K, Y, L, H, F, N, A, R, G, D, E, or I; X37 is I or A; and X41 is V, N, I, F, Y, L, E, or H; (o) an amino acid sequence of SEQ ID NO: 738, wherein X1 is S, F, Q, R, or I; X3 is R, V, or H; X4 is D, E, H, G, or K; X5 is Y, D, G, V, or S; X7 is E or R; X8 is R, F, or V; X12 is E, Y, S, G, N, I, or L; X13 G, K, or N; X14 is F, Y, A, G, Q, L, I, V, H, or K; X16 is S or D; X18 is V or I; X20 is T, Y, F, K, or V; X21 is Q, R, S, or H; X25 is Q or D; X34 is Y, S, G, R, D, F, N, V, A, L, H, I, or K; X41 is N, F, L, I, Y, V, or D; and X47 is R or E; (p) an amino acid sequence of SEQ ID NO: 786, wherein X1 is S, K, or Y; X4 is D or I; X5 is Y or T; X8 is R or K; X13 is G, H, or R; X14 is R or Y; X17 is G, H, D, or Y; X20 is K or Y; X21 is Q or Y; X32 is D, G, S, F, or Y; X34 is Q, Y, L, N, or G; X41 is N, F, I, Y, L, V, or D; and X47 is R, I, or T; IPTS / 128584488.1 Page 17 of 180(q) an amino acid sequence of SEQ ID NO: 811, wherein X1 is S, H, G, L, N, Y, Q, or K; X2 is A or G; X3 is R, H, or Y; X4 is D, Y, F, L, or K; X5 is Y, I, S, A, L, T, or V; X7 is E, Y, I, L, K, S, R, G, A, or Q; X8 is R, L, T, Y, V, or E; X10 is R or Y; X11 is D, H, or R; X12 is E, L, A, Q, I, H, F, or Y; X13 is G, R, or H; X14 is Q, R, F, Y, H, or S; X16 is S, N, or D; X18 is T or V; X20 is Y, Q, K, R, T, S, N, A, or F; X21 is Q, A, G, H, E, or S; X32 is Y, N, V, F, R, L, S, T, Q, or I; and X41 V, F, D, Y, I, S, E, or L; (r) an amino acid sequence of SEQ ID NO: 874, wherein X1 is S or F; X4 is G or D; X7 is E or A; X13 is G or K; X14 is R, H, F, or Y; X17 is H, Y, or G; X20 is Y, K, or R; X32 is Y, R, F, or D; X34 is Q, N, or E; X41 is N, L, Y, F, D, or I; and X47 is R, E; and (s) an amino acid sequence of SEQ ID NO: 896, wherein X1 is S, F, R, Q, G, V; X2 is A or G; X3 is R, Y, K, or H; X4 is D, S, L, or G; X5 is Y, T, or K; X7 is R, K, I, E, G, or A; X11 is D, T, or S; X12 is E, K, S, or V; X13 is H, G, or R; X14 is R, H, I, Q, V, Y, F, N, or A; X16 is S or D; X17 is D, H, Y, F, A, Q, T, R, G, I, E, V, or S; X18 is V, I, or F; X20 is Y, K, T, or S; X21 is Q, A, Y, or G; X32 is F, N, Y, A, S, L, Q, R, I, E, or K; X41 is N, L, D, Y, T, V, K, I, A, E, or F; and X47 is R or E.

[0034] In another aspect a synthetic TNFR1 binding protein comprising: an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively, and D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of SEQ ID NO: 959, wherein X5 is Y or T; X6 is L or I; X7 is R, Q, E, D, or K; X8 is R or Y; X9 is L or Y; and X11 is D or E; (b) D2 comprises an amino acid sequence of SEQ ID NO: 960, wherein X17 is D, G, or H; X20 is E, K, or Y; X21 is G or Q; X23 is L or Y; and X24 is N or I; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 961, wherein X33 is E or S; and X43 is F or Y.

[0035] In certain embodiments, the synthetic TNFR1 binding protein comprises L1 and L2 set forth wherein (i) L1 comprises an amino acid sequence of X12GX14IS; and (ii) L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E or Q; X14 is Y or R; X29 is R, I, E, Q, N, or S; and X32 is D or Y. IPTS / 128584488.1 Page 18 of 180

[0036] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising an amino acid sequence of SEQ ID NO: 958, wherein X5 is Y or T; X6 is L or I; X7 is R, Q, E, D, or K; X8 is R or Y; X9 is L or Y; X11 is D or E; X12 is E or Q; X14 is Y or R; X17 is D, G, or H; X20 is E, K, or Y; X21 G or Q; X24 is N or I; X29 is R, I, E, Q, N, or S; X32 is D or Y; X33 is E or S; X43 is F or Y; and X47 is R, Y, E, or L.

[0037] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively; and (ii) an amino acid of SEQ ID NO: 993, wherein D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of SEQ ID NO: 994, wherein X4 is D, Q, or N; X5 is Y or F; X7 is E, R, H, A, S, or K; X8 is R, Y, L, or V; and X11 is D, K, Q, R, N, S, I, or T; (b) D2 comprises an amino acid sequence of SEQ ID NO: 995, wherein X18 is V, T, N, or I; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 996, wherein X34 is Q, D, E, or N; X40 is A or V; and X41 is L or D. In some embodiments, L1 comprises an amino acid sequence of X12GX14IS and L2 comprises an amino acid sequence of X29GED, wherein X12 is E or A; X14 is Y or L; and X29 is R, I, or L.

[0038] A synthetic TNFR1 binding protein comprising an amino acid sequence of SEQ ID NO: 993, wherein X1 is S, H, R, Y, V, F, or L; X4 is D, Q, or N; X5 is Y or F; X7 is E, R, H, A, S, or K; X8 is R, Y, L, or V; X11 is D, K, Q, R, N, S, I, or T; X12 is E or A, or N; X14 is Y or L; X18 is V, T, N, or I; X29 is R, I, or L; X34 is Q, D, E, or N; X40 is A or V; X41 is L or D; and X47 is R, E, L, Q, or T.

[0039] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 997-1020.

[0040] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively; and (ii) an amino acid of SEQ ID NO: 1021, wherein D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of SEQ ID NO: 1022, wherein X8 is R or Y; and X11 is D, K, or R; (b) D2 comprises an amino acid sequence of SEQ ID NO: 1023; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 1024, wherein X41 is L or D. In some embodiments, L1 IPTS / 128584488.1 Page 19 of 180comprises an amino acid sequence of SEQ ID NO: 1025 and L2 comprises an amino acid sequence of X29GED, wherein X12 is E or A; and X29 is R or I.

[0041] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising an amino acid sequence of SEQ ID NO: 1021, wherein X8 R or Y; X11 is D, K, or R; X12 is E or A; X29 is R or I; X41 is L or D; and X47 is R or E.

[0042] Depending upon the circumstances, it is understood that in a TNFR1 binding protein containing D1 and D3 domains, the D1 domain can be flanked by one or more N- terminal amino acids and / or the D3 can be flanked by one or more C-terminal amino acids, for example, as described herein.

[0043] In another aspect, the disclosure provides synthetic TNFR1 binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 1 or 6-17, 26-48, 53- 73, 78-147, 152-192, 197-271, 276-344, 349-366, 371-403, 408-444, 449-522, 527-562, 567- 602, 607-675, 680-737, 742-785, 790-810, 815-873, 878-895, 900-957, 962-982, and 997- 1020.

[0044] In certain embodiments, the synthetic TNFR1 binding proteins provided herein have a binding affinity for TNFR1 stronger than 10 ^M. The synthetic TNFR1 binding protein can have a binding affinity between about 10 ^M to about 0.1 n ^; about 7.5 ^M to about 0.75 nM; about 5 ^M to about 0.5 nM ^ about 2.5 ^M to about 0.25 nM; about 1 ^M to about 1 n ^; about 0.5 ^M to about 1 nM; about 0.25 ^M to about 1 n ^ ^ ^about 0.10 ^M to about 1 n ^ ^ ^about 75 n ^ to about 1 n ^ ^ ^about 50 n ^ to about 1 n ^ ^ ^about 25 n ^ to about 1 n ^ ^ ^about 10 n ^ to about 1 n ^ ^ ^and about 5 n ^ to about 1 n ^.

[0045] Depending upon the circumstances, the synthetic TNFR1 binding protein has a binding affinity stronger than about 10 ^M, about 7.5 ^M, about 5 ^M, about 2.5 ^M, about 1 ^M, about 0.75 ^M, about 0.5 ^M, about 0.25 ^M, about 0.1 ^M, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, and about 0.1 nM.

[0046] In certain embodiments, D1 is flanked by one or more N-terminal amino acids and / or D3 is flanked by one or more C-terminal amino acids. Depending upon the circumstances, the N-terminus of the first alpha helix comprises an N-terminal extension comprising, e.g., 5, 10, 15, 20, 25, 30, 35, or more amino acids in length. For example, in IPTS / 128584488.1 Page 20 of 180some embodiments, the N-terminal extension has an amino acid sequence comprising that of SEQ ID NO: 1026. In certain embodiments, the C-terminus of the third alpha helix comprises a C-terminal extension comprising, e.g., 5, 10, 15, 20, 25, 30, 35, or more amino acids in length. In some embodiments, the C-terminal extension has an amino acid sequence comprising that of SEQ ID NO: 1029.

[0047] In certain embodiments, the amino acid sequence of the TNFR1 binding protein has at least 65 (e.g., 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5) percent identity to that of SEQ ID NO: 1.

[0048] In certain embodiments, the amino acid sequence of the TNFR1 binding protein has at least 85 (e.g., 90, 95, 96, 97, 98, 99, 99.5) percent identity to that of SEQ ID NO: 9. In certain embodiments, the amino acid sequence of the TNFR1 binding protein has at least 70 (e.g., 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5) percent identity to that of SEQ ID NO: 30. In certain embodiments, the amino acid sequence of the TNFR1 binding protein has an amino acid sequence comprising or according to one or more sequences set forth in Table 12.

[0049] In another aspect, the disclosure provides a multivalent protein comprising a plurality of synthetic TNFR1 binding proteins disclosed herein. The multivalent protein can be bivalent (i.e., binds to two target molecules). The multivalent protein can comprise a first synthetic TNFR1 binding protein and a second synthetic TNFR1 binding protein linked together through at least one linker. The linker can connect a C-terminal amino acid of the first synthetic TNFR1 binding protein to the N-terminal amino acid of the second synthetic TNFR1 binding protein. In certain embodiments, the linker comprises glycine and serine amino acids (e.g., the amino acid sequence GGS). The multivalent protein can have a binding affinity for TNFR1 stronger than the binding affinity of each synthetic TNFR1 binding protein alone.

[0050] In certain embodiments, the multivalent protein comprises a synthetic TNFR1 binding protein having an amino acid sequence, wherein the amino acid sequence comprises an amino acid sequence set forth in Table 12.

[0051] Also provided herein is a pharmaceutical composition comprising a synthetic TNFR1 binding protein or a multivalent protein disclosed herein, and a pharmaceutically acceptable carrier. The synthetic TNFR1 binding protein or multivalent protein can further comprise an effector molecule. IPTS / 128584488.1 Page 21 of 180

[0052] In another aspect, the disclosure provides a method of targeting TNFR1. The method comprises contacting a cell that expresses TNFR1 on its cell surface with a composition comprising a synthetic TNFR1 binding protein or multivalent protein as provided herein. In addition, the disclosure provides a method of modulating TNFR1 activity. The method comprises contacting a cell that expresses TNFR1 on its cell surface with a composition comprising a synthetic TNFR1 binding protein or multivalent protein provided herein. In each of the methods, the synthetic TNFR1 binding protein or the multivalent protein further comprises an effector molecule. Furthermore, depending upon the circumstances, the synthetic TNFR1 binding protein or the multivalent protein inhibits or reduces TNFR1 activity in the presence of a TNFR1 ligand (e.g., TNF ^) relative to TNFR1 activity in the presence of the TNFR1 ligand (e.g., TNF ^) but in the absence of the synthetic TNFR1 binding protein or the multivalent protein.

[0053] In another aspect, the disclosure provides a method of decreasing TNFR1- mediated activity in a subject in need thereof. The method comprises administering to the subject an effective amount of a pharmaceutical composition as provided herein. In another aspect, the disclosure provides a method of treating inflammation in a subject in need thereof. The method comprises administering to the subject an effective amount of the pharmaceutical composition as provided herein. In certain embodiments, the subject is diagnosed as having rheumatoid arthritis, juvenile idiopathic arthritis, plaque psoriasis including pediatric plaque psoriasis, psoriatic arthritis, axial spondylitis including ankylosing spondylitis and non-radiographic axial spondyloarthritis, Crohn’s disease, ulcerative colitis including pediatric ulcerative colitis, uveitis, or Hidradenitis Suppurativa.

[0054] These and other aspects and features of the disclosure are described in the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG.1 is a schematic illustration of an exemplary synthetic protein disrupting the TNFR1 axis by interfering with binding of a TNFR1 ligand (e.g., TNFα) with TNFR1.

[0056] FIGs.2A-2C are circular dichroism (CD) spectra showing the stability profiles of exemplary TNFR1 binding proteins (Reference Miniproteins 8, 11, and 18) disclosed herein over a wavelength range of 200-250 nm during heating and cooling measured at 5oC intervals between temperatures of 24.7 - 95.2 (°C). FIG.2A shows CD spectra of the synthetic TNFR1 binding protein of Reference Miniprotein 18, FIG.2B shows CD spectra of IPTS / 128584488.1 Page 22 of 180the TNFR1 binding protein of Reference Miniprotein 8, and FIG.2C shows CD spectra of the TNFR1 binding protein of Reference Miniprotein 11.

[0057] FIGs.3A-3C are graphs derived from the data in FIGs.2A-2C showing the stability of exemplary TNFR1 binding proteins at 222 nm, between 25 ^C and 95℃. FIG.3A shows an unfolding profile based on CD spectra of the TNFR1 binding protein of Reference Miniprotein 18, which retains a helical signature at 95℃, FIG.3B shows an unfolding profile based on CD spectra of the TNFR1 binding protein of Reference Miniprotein 8, and FIG.3C shows an unfolding profile based on CD spectra of the TNFR1 binding protein of Reference Miniprotein 11. Reference Miniprotein 11 shows an example of cooperative unfolding.

[0058] FIG.4 is a graph showing decrease in TNFα activity (shown as percent neutralization of TNFα) in human embryonic kidney cells pretreated for thirty minutes with one of eight different concentrations (between 0.0001 and 1.000 nM) of an unrelated binding protein (control that does not bind TNFR1),an exemplary TNFR1 binding protein (Reference Miniprotein 19) or an exemplary bivalent miniprotein (Reference Miniprotein 32) prior to the addition of TNFα to the cell culture.

[0059] FIG.5 is a graph showing decrease in cell death in vitro after exposure to TNFα in presence of exemplary TNFR1 binding proteins disclosed herein or an unrelated binding protein (control that does not bind TNFR1). The viability of mouse L929 cells was measured by luminescence (RU, y-axis) in cells pretreated with one of seven different concentrations (between 0.001 and 1,000 nM) of a control nonbinding protein, an exemplary monovalent TNFR1 binding protein (Reference Miniprotein 23) or an exemplary bivalent TNFR1 binding protein (Reference Miniprotein 32).

[0060] FIG.6 is a survival profile graph showing the prevention TNFα-mediated death in vivo using treatment with exemplary TNFR1 binding proteins. The y-axis shows percent survival of C57 / BL6 mice up to 48 hours (x-axis) after intravenous injection of TNFα before, concomitant with, or after exposure to unrelated binding protein (control), an exemplary TNFR1 binding protein, or a TNFα antibody. Mice were injected with a dose of TNFα typically considered to be lethal. The TNFα was injected into all mice, and groups were divided into one of four conditions: (i) pretreatment with control miniprotein 20 minutes prior to TNFα injection; (ii) pre-treatment with a TNFα antibody 60 minutes prior to TNFα injection; (iii) treatment with an exemplary monovalent TNFR1 binding protein (Reference IPTS / 128584488.1 Page 23 of 180Miniprotein 23) 20 minutes prior to, the same time as, or 20 minutes after TNFα injection; and (iv) treatment with a bivalent TNFR1 binding protein (Reference Miniprotein 32) 20 minutes prior to, the same time as, or 20 minutes after TNFα injection. DETAILED DESCRIPTION

[0061] The disclosure is based, in part, upon the discovery of synthetic TNFR1 binding proteins that specifically bind TNFR1 and modulate the TNFR1 axis, for example, by preventing ligand binding and reducing or eliminating downstream TNFR1-signaling activity. The TNFR1 binding proteins described herein have specific binding activity for TNFR1, have the ability to reduce downstream TNFR1-mediated signaling activity, and are, among other things, thermally and chemically stable, resistant to oxidation, resistant to protease degradation, deamination, and glycosylation, and have mM-level solubility. The binding proteins can be monovalent, bivalent, or multivalent and can also be conjugated (e.g., via chemical conjugation or as a fusion protein) to an effector molecule.

[0062] Accordingly, the present disclosure provides, among other things, synthetic TNFR1 binding proteins, methods of making such binding proteins, and methods of using such proteins to treat a disease or disorder mediated by the TNFR1 axis. I. DEFINITIONS

[0063] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. For example, nomenclatures utilized in connection with, and techniques of, e.g., polypeptide and polynucleotide chemistry and synthesis, molecular and cellular biology, protein biology and biochemistry, immunology, etc. described herein are those well-known and commonly used in the art.

[0064] As used herein, the singular forms “a,” “an” and “the” include plural referents unless context clearly dictates otherwise. Thus, for example, in some embodiments, reference to, e.g., a synthetic TNFR1 binding protein includes a single binding protein, a plurality of synthetic binding proteins, etc.

[0065] As used herein, the expression “and / or” in connection with two or more recited objects includes individually each of the recited objects and the various combinations of two or more of the recited objects, unless otherwise understood from the context and use. IPTS / 128584488.1 Page 24 of 180

[0066] Where the use of the term “about” is before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0067] As used herein, the phrase “alpha helix cap residue” refers to an amino acid located at the N-terminus, but not within, a set of amino acids that together form an alpha helical secondary structure in a polypeptide.

[0068] As used herein, the phrases “solvent accessible residue” and “solvent accessible amino acid” refer to an amino acid that, when disposed in a folded molecule (e.g., in its a tertiary conformation) and in a solvent, is characterize in that the amino acid is at least partially accessible or exposed to the solvent. Solvent accessible amino acids can be determined using a variety of approaches including, e.g., Rosetta software suite, Neighbor Count, and Neighbor vector algorithms (Durham et. al. (2009) J. MOL. MODEL.15(9): 1093-108).

[0069] As used herein, the phrase “conservative substitution” refers to a substitution with a structurally and / or functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S) and Threonine (T); 2) Aspartic Acid (D) and Glutamic Acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W). Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), or the PAM substitution:p matrix (e.g., the PAM 250 matrix). In certain embodiments, a binding protein of the disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions relative to a reference amino acid sequence.

[0070] As used herein, the phrase “corresponding to” designates a position / identity of an amino acid or a nucleic acid in a polymeric molecule such as an amino acid in an amino acid sequence or a nucleic acid in a nucleic acid sequence. It is understood by the skilled artisan that such amino acids or nucleic acids in such a polymer are often designated using a canonical numbering system based on a reference related polymer, so that, for example, an amino acid in a first polymer “corresponding to” position seven in the reference amino acid, for example, need not actually be the seventh amino acid in the first polymer. Those IPTS / 128584488.1 Page 25 of 180of ordinary skill in the art are aware of methodology to identify “corresponding” amino acids or nucleic acids between two molecules (e.g., a polymer and a reference polymer), including, such as, commercially available algorithms, databases, or other information given context regarding particular polymers.

[0071] As used herein, the term “domain” refers to a region or segment of a given synthetic binding protein disclosed herein, and can include one or more structural features (e.g., amino acid, primary, structure or secondary structure features) and / or one or more functional features (e.g., binding properties). In the context of secondary structure, a “structural domain” can be an uninterrupted linear sequence that adopts a single type of secondary structure, for example, ten continuous amino acid residues that are all part of the same alpha helix structure. In the context of binding, a “binding domain” can be a discontinuous portion of the overall amino acid sequence that facilitates chemical interactions with a target molecule or indirectly stabilizes such interactions.

[0072] As used herein, the term "paratope" refers to a specific region of a binding molecule (e.g., a binding protein, e.g., a TNFR1 binding protein) that recognizes and binds an epitope of a target molecule (e.g., TNFR1). A paratope of a given binding molecule typically comprises 4-20 (e.g., 4-10, e.g., 4-8) amino acids that are solvent accessible and in close proximity in three-dimensional space.

[0073] As used herein, the phrase “effective amount” refers to the amount of an active agent (e.g., a synthetic TNFR1 binding protein disclosed herein) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route.

[0074] As used herein, the term “effector” refers to a molecule or molecular entity that confers one or more particular characteristics on itself or another molecule or molecular entity with or to which it is associated. For example, an effector may include a synthetic binding protein (e.g., a miniprotein, or something other than a miniprotein that is associated with a synthetic TNFR1 binding protein disclosed herein (e.g., via a covalent linkage), such as a detectable label (e.g., visualizable or otherwise measurable such as by fluorescence or radiolabel detection), small molecule, nanoparticle (e.g., a lipid nanoparticle, a polymer nanoparticle, etc.), polynucleotide (e.g., an aptamer, an siRNA, an shRNA, an oligonucleotide, etc.), a radionuclide, etc. An effector may be a synthetic binding protein IPTS / 128584488.1 Page 26 of 180(e.g., a monovalent synthetic binding protein linked to a TNFR1 binding protein disclosed herein to create a bivalent synthetic protein where one or both of the proteins causes a change, e.g., in a cellular function, e.g., in a disease state, etc.).

[0075] As used herein, the term “loop” refers to (i) a structure (e.g., polypeptide) that connects two structural domains (e.g., a loop may be disposed between two alpha helices or between an alpha helix and a beta sheet in a given synthetic TNFR1 binding protein) and / or (ii) a structure (e.g., peptide) present at the N- and / or C-terminal end of a given monovalent synthetic binding protein.

[0076] As used herein, the term “linker” refers to a structure (e.g., a polypeptide linker, or a chemical crosslinker (e.g., a homobifunctional or a heterobifunctional cross linking agent) between two molecules (e.g., two synthetic TNFR1 binding proteins disclosed herein) or between, e.g., a synthetic TNFR1 binding protein and an effector, wherein each of the entities that are linked is covalently linked to one another.

[0077] As used herein, the terms / phrases “synthetic binding protein,” “synthetic miniprotein,” and “miniprotein” are used interchangeably, and refer to a polypeptide between about 25 to about 100 amino acids in length, e.g., about 30 to about 90 amino acids, about 35 to about 65 amino acids, or about 40 to about 50 amino acids in length that are capable of binding to a given target, e.g., TNFR1 with a desired binding affinity (e.g., stronger than 10 µM).

[0078] As used herein, the phrase “percent identity” and “% identity” refers to the extent to which two sequences (e.g., a polypeptide) have the same amino acid or nucleotide at the same positions in an alignment. The percent identity between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. It is contemplated that a reference sequence can be an amino acid sequence corresponding to an entire TNFR1 binding protein or a portion thereof. A reference sequence may be an amino acid sequence that corresponds to a particular domain or domains (e.g., an alpha helix, a loop region) or a combination of domains (e.g., a combination of a loop and an alpha helix). Alignment for purposes of determining percent sequence identity (e.g., amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such IPTS / 128584488.1 Page 27 of 180as BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. For a discussion of basic issues in searching sequence databases, see Altschul et al., (1994) NATURE GENETICS 6:119-129, which is incorporated by reference herein. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0079] As used herein, the term “synthetic” refers to a molecule that is (i) not naturally occurring, (ii) not present in nature, (iii) does not comprise entirely natural components, or (iv) a combination of any one of (i), (ii) and (iii). For example, a synthetic peptide does not exist naturally, and is produced or otherwise modified by human intervention, such as techniques including recombinant or cell-free synthesis, and / or the peptide may comprise one or more non-naturally occurring amino acids.

[0080] As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0081] As used herein, the phrase “pharmaceutically acceptable carrier” as used herein refers to an agent (e.g., excipient, carrier, buffer, etc.) suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Standard pharmaceutical carriers may include, for example a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020).

[0082] As used herein, “treat”, “treating”, and “treatment” refer to the treatment of a disease, disorder, or symptom or manifestation of such in a subject, e.g., in a human. This IPTS / 128584488.1 Page 28 of 180includes: (a) preventing a disease or disorder, (b) inhibiting the disease, disorder, etc., i.e., slowing or arresting its progress or development; and (b) relieving the disease, disorder, etc., i.e., causing regression of the disease state. As used herein, “prevent”, “preventing” and “prevention” refer to causing a disease, disorder, or symptom or manifestation of such not to occur for at least a period of time in at least some subjects.

[0083] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.

[0084] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps. Similarly, throughout the description, where compositions are described as consisting essentially of specific components, or where processes and methods are described as consisting essentially of specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist of the recited components, and that there are processes and methods according to the present disclosure that consist of the recited processing steps.

[0085] Throughout the text, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0086] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and / or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and IPTS / 128584488.1 Page 29 of 180concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and any invention provided herein. For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of any invention described and depicted herein.

[0087] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of any invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of any invention disclosed herein.

[0088] It should be understood that the expression “at least one of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use.

[0089] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context

[0090] It should be understood that the order of steps or order for performing certain actions is immaterial so long as disclosed invention(s) remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0091] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc. II. TUMOR NECROSIS FACTOR RECEPTOR 1 (TNFR1)

[0092] Provided herein are synthetic binding proteins (also referred to as miniproteins), that bind TNFR1. TNFR1 is a monomeric transmembrane receptor that binds TNF ^ as well as other ligands, including, lymphotoxin- ^. TNFR1 belongs to a family of receptors known as the TNF receptor (TNFR) superfamily. The TNFR superfamily is a group of type I transmembrane proteins, with a carboxy-terminal intracellular domain and an amino-terminal IPTS / 128584488.1 Page 30 of 180extracellular domain characterized by a common cysteine rich domain (CRD). The gene structure of TNFR1 extends over 10 exons separated by 9 introns (Fuchs, et al. (1992) GENOMICS 13:219; Santee, et al. (1996) J. BIOL. CHEM. 35:21151).

[0093] As depicted schematically in FIG.1, soluble TNF ^ is believed to bind to TNFR1 as a homotrimer, with three molecules of soluble TNF ^ arranged in a homotrimer that binds to clustered TNFR1 receptor monomers (see, e.g., McMillan, et al. (2021) NAT. COMMUN. 12:582). Without wishing to be bound by theory, current understanding suggests a mechanism whereby a soluble TNF ^ homotrimer binds to pre-clustered TNFR1 dimers in the pre-ligand assembly domain (PLAD) and subsequently allows TNFR1 trimers to form (see, e.g., McMillan, et al. (2021) supra). Thus, binding of a TNF ^ homotrimer to cell- membrane localized TNFR1 is believed to cause further receptor clustering (e.g., trimerization), leading to cytosolic phosphorylation and downstream signaling, including to cell death pathways (see, e.g., van Loo and Bertrand (2022) NAT. REV. IMMUNOL. 23: 289).

[0094] TNFR1 is also known as CD120a (see, e.g., GenBank accession number X55313 for human TNFR1) and has a molecular weight of approximately 55 kilodaltons (kDa). TNFR1 is ubiquitously expressed throughout the human body and mediates pro-inflammatory and pro-apoptotic responses (Sethi et al. (2021) NAT. METAB. 3: 1302). For instance, and as shown in FIG.1, it is believed that ligand binding (e.g., TNFα) to TNFR1 activates a cytoplasmic death domain that binds to adaptor proteins TNFR1-associated death domain (TRADD) and Fas-associated death domain (FADD) and triggers apoptosis (see, e.g., van Loo and Bertrand (2022) supra). In addition, it is believed that TNF-induced TNFR1 signaling can activate classical nuclear factor kappa B (NFkB) proinflammatory signaling through TRADD (see, e.g., van Loo and Bertrand (2022) supra). In this pathway, TRADD recruits and interacts with TRAF2, which can activate various downstream MAPKs and JNK molecules (see, e.g., Shi et al. (2018) FRONT. IMMUNOL. 9: 1849).

[0095] Overactivation of TNFR1 (e.g., by ligand binding such as by TNF ^) can be systemically toxic. For example, a TNFR1-selective antagonistic mutant (RlantTNF) was found to selectively and specifically inhibit TNFR1-mediated signaling (see, e.g., Shibata et al. (2008) CYTOKINE 44(2): 229) in murine acute hepatitis models and a murine collagen- induced arthritis model resulting in improvements of one or more normally-elevated enzymes or cytokines. The beneficial effect of selective TNFR1 inhibition has been further supported by studies with a negative dominant TNF mutant (XPro1595) capable of forming inactive IPTS / 128584488.1 Page 31 of 180complexes with soluble TNF ^, thus selectively inhibiting pro-inflammatory action mediated by TNFR1 while preserving innate immunity to infections (see, e.g., Olleros et al. (2009) J. INFECT. DIS. 199 (7): 1053-63).

[0096] Given the role that TNFR1-mediated signaling in believed to play in certain autoimmune diseases, such as, for example, rheumatoid arthritis, psoriatic arthritis, plaque psoriasis, Crohn’s disease, and ulcerative colitis, inhibition of TNFR1 itself, rather than of TNF ^, has gained increasing attention. Many of the common approved therapeutics bind to TNF ^ in an attempt to reduce binding to TNFR1, but, in addition to cost and concerns about long-term safety with use of biologics, such antibodies are not always be effective at treating autoimmune diseases in all patients. See, e.g., Rubbert-Roth et al. (2009) ARTHRITIS. RES. THER. 11 (Suppl 1): S1.

[0097] Use of TNF ^ ^binding agents and TNFR1-binding agents (e.g., divalent antibodies, e.g., monovalent antibodies) or fragments thereof (e.g., scFv, dAb, Fab, nanobodies, etc.) can include risks of pro-inflammatory reactions, including cytotoxicity and apoptosis, and can have relatively short half-lives, requiring repeated administration, which can increase risk of toxicity (see, e.g., Toussirot and Aubin (2016) supra; see also Li, et al. (2017) supra). Thus, there remains a need for effective and / or improved anti-TNFR1 binding agents. III. TNFR1 BINDING PROTEINS

[0098] The disclosure provides synthetic TNFR1 binding proteins, and methods of identifying, making, characterizing, formulating, and using such TNFR1 binding proteins. The synthetic TNFR1 binding proteins have several advantages over small molecule and large molecule (e.g., biologics) based therapeutics. For example, small molecule therapeutics may suffer from off-target activities and / or may have long half-lives, which can risk negative impact on one more organs or organ systems, such as kidneys (see, e.g., Li, et al. (2017) supra). Similarly, large molecules such as biologics are expensive to produce, are challenging to produce in uniform batches of drug substance, and can be challenging to formulate, transport, store, and administer to subjects. Furthermore, in some instances, antibodies to TNFR1 have been found to act as agonists in vivo (see, e.g., Richter et al., (2021) FRONT. IMMNOL. 12: 705485 citing to Fischer et al. (2020) FRONT. CELL DEV. BIOL. 8:401). The synthetic TNFR1 binding proteins disclosed herein avoid such disadvantages as they have high specificity to TNFR1, can be engineered to have desired IPTS / 128584488.1 Page 32 of 180pharmacodynamic and pharmacokinetic properties (e.g., a desirable circulating half-life in plasma), reduced immunogenicity (e.g., do not elicit an immune response against a therapeutic drug, e.g., a therapeutic antibody), are chemically and thermally stable, and are resistant to protease degradation, deamination and post translational modification (e.g., glycosylation), and are stable in different redox environments.

[0099] A synthetic TNFR1 binding protein described herein can have mM-level solubility. In some embodiments, the TNFR1 binding protein can have a solubility greater than 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, or 80 mg / mL in an aqueous solution.

[0100] The TNFR1 binding proteins described herein designed to specifically bind TNFR1, and preferably bind TNFR1 over TNFR2. Preferably, the binding proteins do not bind TNFR2. In some embodiments, the disclosure provides a means for specifically binding TNFR1 over TNFR2. Depending upon the circumstances, the TNFR1 binding proteins bind TNFR1 with a binding affinity greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold greater than TNFR2.

[0101] The synthetic TNFR1 binding proteins disclosed herein can be conjugated (e.g., chemically conjugated to or are fusion proteins) to one or more effector molecules. For the example, the TNFR1 binding proteins described herein can be bound to one or more other synthetic binding proteins, which can be the same or different. The resulting proteins can be monovalent, bivalent, or multivalent. For example, a first TNFR binding protein described herein can be conjugated to a second TNFR binding protein that can be the same as or different from the first TNFR binding protein. The resulting molecule is bivalent (see Example 6, for an exemplary bivalent TNFR1 binding protein). Alternatively, a first TNFR1 binding protein described herein can be conjugated to a second binding protein that binds a different target of interest such as, e.g., TNFR2 (e.g., to eliminate or reduce all TNF-receptor signaling), serum albumin (e.g., to extend serum half life), a tissue specific surface protein (e.g., to target TNFR1 inhibition only to diseased tissue such as joints or gut), etc. The resulting molecule is bivalent and can bind TNFR as well as the second target molecule.

[0102] In one aspect, the disclosure provides a synthetic TNFR1 binding protein that comprises: (a) an amino acid sequence from 30 amino acids to 95 amino acids in length; IPTS / 128584488.1 Page 33 of 180(b) a net negative charge in phosphate buffered saline (PBS); (c) a binding affinity for TNFR1 stronger than 10 ^M; and (d) a stability profile such that the protein (i) retains at least 90% binding affinity to TNFR1 upon cooling to room temperature after thermal denaturation at 95oC in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to TNFR1 after incubation for 16 hours at 37oC of incubation in PBS relative to the protein under the same conditions prior to incubating; and / or (iii) retains at least 90% binding affinity to TNFR1 in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

[0103] In another aspect, the disclosure provides a synthetic TNFR1 binding protein, the binding protein comprising: (a) an amino acid sequence from 30 amino acids to 95 amino acids in length; (b) a net negative charge in PBS; (c) a binding affinity for TNFR1 stronger than 10 ^M; (d) at least three alpha helices; (e) at least two amino acid loops, where a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., a N-terminal amino acid) of a second alpha helix, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., a C-terminal amino acid) of the second alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a third alpha helix; and (f) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one, two or three of the three alpha helices.

[0104] Synthetic TNFR1 binding proteins of the present disclosure may include naturally- occurring or non-naturally occurring amino acids. It is understood that certain amino acids may have and / or take on different characteristics (e.g., hydrophobic, hydrophilic, neutral, etc.) depending upon the context (e.g., macro and / or micro-environment including, but not limited to surrounding amino acids, environmental conditions such as solvent type, pH, etc.). Various terms and phrases may be used herein to describe, identify, and / or characterize amino acids. In addition, a single amino acid, at any given time, may have more than one characteristic or identity. Depending upon the context, a hydrophobic amino acid may be selected from alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, tryptophan, tyrosine, lysine, and arginine. A hydrophilic IPTS / 128584488.1 Page 34 of 180amino acid may be an amino acid selected from cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, tryptophan, and tyrosine. A charged amino acid may be an amino acid selected from arginine, histidine, lysine, aspartic acid (aspartate), and glutamic acid (glutamate). A positively-charged amino acid may be an amino acid selected from arginine, histidine, and lysine. A negatively- charged amino acid may be an amino acid selected from aspartic acid (aspartate) and glutamic acid (glutamate). An uncharged or neutral amino acid may be selected from alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine.Synthetic TNFR1 binding proteins of present disclosure have a primary structure comprising certain key features. For example, synthetic TNFR1 binding proteins can have amino acid sequences that include various combinations of hydrophobic and solvent accessible amino acids organized into certain domains. Primary structures (i.e., amino acid sequences) of the synthetic TNFR1 binding proteins will have a combination of one or more types (e.g., hydrophobic, e.g., solvent accessible) amino acids.

[0105] In certain embodiments, the synthetic TNFR1 binding protein comprises one or more of the following features (a) the first, second, and / or third alpha helix each contains at least one hydrophobic amino acid; (b) the first, second, and / or third alpha helix each contains at least two or three hydrophobic amino acids; (c) the first, second, and / or third alpha helix each contain at least one or two solvent accessible amino acids; (d) the first and / or second loop contains at least one hydrophobic amino acid, or (e) the binding protein comprises any combination of elements selected from (a), (b), (c), and (d).

[0106] Similarly, it is contemplated that the TNFR1 binding can comprise one or more of the following features: (a) the second and third alpha helix each contains at least two hydrophobic amino acids; (b) the first, second, and third alpha helix each contains at least one solvent accessible amino acid; (c) the first, second, and third alpha helix each contain at least two hydrophobic and one solvent accessible amino acids; (e) the second and third alpha helix each contains at least four solvent accessible amino acids; and / or (f) the first loop contains at least one hydrophobic amino acid.

[0107] In each of the foregoing aspects, the synthetic TNFR1 binding protein comprises one or more of the following features: (a) free of tryptophan amino acids; (b) free of methionine amino acids; (c) free of lysine amino acids; (d) does not comprise an unpaired IPTS / 128584488.1 Page 35 of 180cysteine amino acid when cysteine amino acids are present in the protein; (e) free of glycosylation sites; (f) free of protease cleavage sites; and (g) soluble up to at least 1 mM in PBS at 4oC for one month. Preferably, the TNFR1 binding sites are designed to be free of glycosylation sites, for example, free of peptide sequences that are substrates for glycosylation (e.g., N-X-S / T, which can be a substrate for an oligosaccharyltransferase (OST) complex). Alternatively or in addition, the TNFR1 binding sites are designed to be free of protease cleavage sites, for example, free of peptide sequences that are substrates for proteases (e.g., L-X-R-R, which can be a substrate for a Kexin / KEX2 protease). Similarly, the TNFR1 binding proteins may also be designed to be free of other protease cleavage sites for other proteolytic enzymes such as trypsin, chymotrypsin, elastase, subtilisin, etc.

[0108] Depending upon the circumstances, the N-terminus of the first alpha helix is preceded by one or more N-terminal amino acids and / or the C-terminus of the third alpha helix is followed by one or more C-terminal amino acids. It is contemplated that the synthetic TNFR1 binding protein can comprises from 35 amino acids to 85 amino acids in length, from 35 amino acids to 75 amino acids in length, from 35 amino acids to 65 amino acids in length, from 35 amino acids to 55 amino acids in length, from 35 amino acids to 50 amino acids in length, from 40 amino acids to 85 amino acids in length, from 40 amino acids to 75 amino acids in length, from 40 amino acids to 65 amino acids in length, from 40 amino acids to 55 amino acids in length, or from 40 amino acids to 50 amino acids in length. In certain embodiments, the synthetic TNFR1 binding protein of claim comprises 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0109] In certain embodiments, a miniprotein of the disclosure can have an N- and / or C- terminal extension. These extensions can serve a variety of purposes including for use in conjugation and / or assays. An N-terminal extension and / or a C-terminal extension can each comprise a polypeptide linked to the miniprotein by a linker or by a peptide bond. An N-terminal extension and / or a C-terminal extension can each independently comprise or consist of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. Addition of an N- and / or C-terminal linker to a TNFR1 binding protein of the disclosure would extend the overall length of the TNFR1 binding protein by the total number of amino acids in the N- and / or C-terminal extensions. TNFR1 binding proteins of the disclosure are still able to bind TNFR1, including with affinities as provided herein. IPTS / 128584488.1 Page 36 of 180

[0110] An N-terminal extension and / or a C-terminal extension can be or comprise one or more tags (e.g., a myc tag, a hemagglutinin (HA) tag, etc.). Sequences of myc tags and HA tags are known to those of skill in the art. An exemplary HA tag sequence is set forth in SEQ ID NO: 1030 (YPYDVPDYA). An exemplary myc tag sequence is set forth in SEQ ID NO: 1031 (EQKLISEEDL).

[0111] An N-terminal extension and / or a C-terminal extension can be a linker or can comprise a linker. A linker can be an ordered or disordered linker. A linker can be a peptide linker, such as set forth in SEQ ID NOs: 1028 (GSGSGGGSGGSSGGS) and / or SEQ ID NO: 1032 (SSGGSSSSGSGSGSGGGGGGS). Combinations of tags and linkers can be used to make up N- and / or C-terminal extensions as disclosed herein.

[0112] For example, in certain embodiments, a miniprotein of the disclosure may further comprise an N-terminal extension comprising or consisting of a polypeptide having an amino acid sequence of SEQ ID NO: 1026 (KDNSSTIEGRYPYDVPDYALQAGSGSGGGSGGSSGGS) and / or a C-terminal extension comprising or consisting of a polypeptide having an amino acid sequence of SEQ ID NO: 1029 (SSGGSSSSGSGSGSGGGGGGSEQKLISEEDL). An N-terminal extension can comprise a polypeptide having an amino acid sequence of KDNSSTIEGRYPYDVPDYALQA (SEQ ID NO: 1027) and / or GSGSGGGSGGSSGGS (SEQ ID NO: 1028). In certain embodiments, polypeptides of SEQ ID NOs: 1027 and 1028 can be linked by a peptide bond between the C-terminal alanine of SEQ ID NO: 1027 and the N-terminal glycine of SEQ ID NO: 1028 to form an N-terminal extension as set forth in SEQ ID NO: 1026. A C-terminal extension can comprise a polypeptide having an amino acid sequence of SEQ ID NO: 1031 and / or GSGSGGGSGGSSGGS (SEQ ID NO: 1032). The polypeptides of SEQ ID NOs: 1032 and 1031 can be linked by a peptide bond between the C-terminal glycine of SEQ ID NO: 1032 and the N-terminal glutamic acid of SEQ ID NO: 1031 to form an N-terminal extension as set forth in SEQ ID NO: 1029.

[0113] TNFR1 binding proteins of the disclosure can have an N- and C-terminal extension, such as set forth in SEQ ID NOs: 1026 and 1029 and still bind TNFR1. By way of non-limiting example, a TNFR1 binding protein (e.g., Reference Miniprotein 51 of SEQ ID NO: 997) that is being tested in a cell-based assay such as a yeast display (see, e.g., are capable of binding TNFR1) can have an N- and C-terminal extension, such as set forth in IPTS / 128584488.1 Page 37 of 180SEQ ID NO: 1033, or 1034-1056. Such TNFR1 binding proteins bind to TNFR1 with at least nM affinity as measured by yeast-display assay.

[0114] A synthetic TNFR1 binding protein of the disclosure may have an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more or 100% identity with or to a reference sequence or component thereof, wherein the reference sequence is selected from any of SEQ ID NOs: 1, 6-17, 26-48, 53-73, 78-147, 152-192, 197-271, 276-344, 349-366, 371- 403, 408-444, 449-522, 527-562, 567-602, 607-675, 680-737, 742-785, 790-810, 815-873, 878-895, 900-957, 962-982, 983-989, 997-1020, and portions (e.g., domains) thereof. In some embodiments, a synthetic TNFR1 binding protein may have an amino acid sequence comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more or 100% identity with or to a reference sequence or component thereof selected from any of SEQ ID NOs: 2-5, 18-25, 49-52, 74-77, 148-151, 193-196, 272-275, 345-348, 367-370, 404-407, 445-448, 523-526, 563-566, 603-606, 676-679, 738-741, 786-789, 811-814, 874- 877, 896-899, 958-961, 990-992, 993-996, and 1021-1024, 1033-1056, and portions thereof.

[0115] By way of non-limiting example, a structural arrangement in a synthetic TNFR1 binding protein may be depicted as N-Hn1Ln2Hn3Ln4Hn5-C, where H is an alpha helix, L is a loop, each of n1-n5 represents an integer indicating the number of amino acids in that structural domain, N and C represent N-terminal and C-terminal domains, respectively. The number of H amino acids does not have to be the same across helices, for example, n1, n3, and n5may be, but do not have to be, the same numbers. Similarly, n2and n4may be, but do not have to be, the same number. For example, an exemplary formula of a synthetic TNFR1 binding protein may comprise N-H10L5H12L4H11 as depicted pictorially below denoting the amino acids in a helix domain (H) or a loop domain (L): N-HHHHHHHHHHLLLLLHHHHHHHHHHHHLLLLHHHHHHHHHHH-C.

[0116] Any given H domain (e.g., Hn1) in an alpha helix that is part of a synthetic TNFR1 binding protein may independently contain between about 4 amino acids and about 20 amino acids in length. An H domain may independently comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length. IPTS / 128584488.1 Page 38 of 180

[0117] Loops disposed between alpha helices may also be of the same or different lengths. Each loop may independently comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 amino acids in length.

[0118] Each alpha helix may comprise the same number of amino acids in each of its H domains or different numbers of amino acids in length in reference to the primary structure of each helical region. That is, in some synthetic TNFR1 binding proteins each H domain in the binding protein is the same length. In some synthetic TNFR1 binding proteins one or more H domains has a different length relative to other H domains in the binding protein. In some embodiments, an H domain has zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven or more conserved amino acids (e.g. relative to other TNFR1 binding proteins).

[0119] A person skilled in the art can determine which amino acids of a given sequence constitute a loop or a helix (see, e.g., Mirdita, et al. (2022) NAT. METHODS (19): 679-682).

[0120] In an exemplary TNFR1 binding protein disclosed herein, the binding protein comprises at least three alpha helices, and at least two loops (a first loop and a second loop), wherein the first loop has a first amino acid sequence that connects a terminal amino acid (e.g., a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., a N-terminal amino acid) of a second alpha helix, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., an C-terminal amino acid) of the second alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a third alpha helix.

[0121] It is contemplated that, when the TNFR1 binding protein comprises an alpha helix, the alpha helix may be preceded by an alpha helix cap amino acid. An alpha helix cap residue precedes the first amino acid in an alpha helix but is not part of the alpha helix itself. It may be part of a loop or it may be, e.g., an N-terminal amino acid that is not a loop, per se, when the synthetic TNFR1 binding protein is monovalent, but could be present in a loop in a multivalent molecule. A synthetic TNFR1 binding protein comprising three alpha helices, can have three alpha helical cap residues, one capping each alpha helix. Each alpha helix cap amino acid may be independently selected from serine, threonine, aspartate, and asparagine.

[0122] A synthetic TNFR1 binding protein may have one of several consensus sequence structures. Consensus sequences will generally have certain “fixed” amino acid positions IPTS / 128584488.1 Page 39 of 180as well as those that can be varied, such as by changing to another amino acid. Sometimes changing amino acids at certain positions can alter the function of the synthetic binding protein by increasing or decreasing affinity for the target (i.e., TNFR1). However, all the binding proteins disclosed herein, although having different primary structures have a minimal “threshold” binding affinity to TNFR1. In some embodiments, a threshold binding affinity may be greater than about 10 ^M, about 1 ^M, about 100 nM, about 10 nM, or about 1 nM.

[0123] A synthetic TNFR1 binding protein disclosure may be represented according to a formula shown as one or more domains, wherein each domain optionally has one or more conserved amino acid residues and / or a particular structure (e.g., loop, e.g., helix). For example, an exemplary synthetic TNFR1 binding protein comprises an amino acid sequence arranged in a primary structure of: D1-L1-D2-L2-D3 (Formula I) where D1, D2 and D3 represent alpha helices and L1 and L2 represent loops connecting the alpha helices. The amino acid sequence of the starting “parental” protein and exemplary consensus sequences for various miniproteins developed in Examples 1-4 are set forth in TABLE 1. Bold residues represent helices and correspond to D1, D2, and D3 in order along a given consensus sequence. Exemplary consensus sequences for D1, D2, and D3 for each miniprotein are set forth in TABLE 2A. Positions for each variable amino acid along the length of a consensus sequence as set forth in TABLE 1 are set forth in TABLE 2B. IPTS / 128584488.1 Page 40 of 180LQX1LV87L18X10D2I8L1DLL8Q1LDDEa2 X1 X XX L 1 2 N G o0X 6 7XLQ1D6DIX7X0LQ92n2711X1X812X1XLQ1X2 G XiX LXVSI461X 0 I Q 6XLELDL ma7I 1 XD62X41021XV781LD14 sX1XX3I1411XLIVX73X1LIV41XXYNLeSI3 X X 4 11 S S Q taR1 2 3 1 XX271X3I4I4GEc3X121X1XX3SI1XXS1X1X1 L idX1X11211X4 1 I 2 GXGVYni 2 11 X X 21X1X411X2121SItxeX1c1X01111X3101X311X X Yen1RXLX8LX8RDLRX X 1 X1111G2tLEDL8XERLXRXR1XdeuRqLX7X78X8XRLX7D R 9 L 1eze8SXX X 7 7 8 XRL7XX88X1XisEL5L5XLXLXEL5R L X 7 RciluLs5X X 5 5 L X7X5X7XXLL8an X4X4XX4X45X4XLY4 6 5 X ties4X3 3 X X 4 3 4X3X5X4ELdnoRXA2X23X3XXRX2XRX2X X Y nC1XX1XX1A A A X A XDRRADRaX1X1X1X1X1X1XAS1XAS,de:nOilNrD3 3 3 6 8 6 1 4 61eI2 dQ56506763787187898853992901nuES,dedl12 3 o1se .1.14 5 6 7 8 9 1.1 1 1 1 1 1 . B*i4 4 4.4.4.4.4.4.456 6rerSerSerSerSerSerSerSerSerSer r rSeSeSeS IPTS / 128584488.1 Page 42 of 180TABLE 2A. Exemplary Miniprotein Domain Consensus Sequences Category SEQ ID NO: Consensus Sequence ARX4YX6X7X8LRX11IPTS / 128584488.1 Page 43 of 180Category SEQ ID NO: Consensus Sequence Di 3 S 47 348EX34AVX37DYAX41DYIX45IPTS / 128584488.1 Page 44 of 180Category SEQ ID NO: Consensus Sequence Di 3 S 416 789EX34AVIDYAX41DYIEIPTS / 128584488.1 Page 45 of 180TABLE 2B. Exemplary Miniprotein Variable Substitutions Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position , ,IPTS / 128584488.1 Page 46 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position , , , S, , , ,IPTS / 128584488.1 Page 47 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position , L, , ,IPTS / 128584488.1 Page 48 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: PositionIPTS / 128584488.1 Page 49 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position , ,IPTS / 128584488.1 Page 50 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: PositionIPTS / 128584488.1 Page 51 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position D,IPTS / 128584488.1 Page 52 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position ,IPTS / 128584488.1 Page 53 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: PositionIPTS / 128584488.1 Page 54 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position IIPTS / 128584488.1 Page 55 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position , NIPTS / 128584488.1 Page 56 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position N AIPTS / 128584488.1 Page 57 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position ,IPTS / 128584488.1 Page 58 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position N, F,IPTS / 128584488.1 Page 59 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position , , EIPTS / 128584488.1 Page 60 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position L,IPTS / 128584488.1 Page 61 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: PositionIPTS / 128584488.1 Page 62 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position I,IPTS / 128584488.1 Page 63 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position , ,IPTS / 128584488.1 Page 64 of 180Amino Acid Consensus Series Consensus Amino Acids Position SEQ ID NO: Position A. Pa

[0124] A synthetic TNFR1 binding protein can bind to its target via a paratope. A paratope of the disclosure can be represented by paratope consensus sequence. Consensus sequences sometimes have certain “fixed” amino acid positions as well as those that can be varied, such as by changing an amino acid at a certain position to another amino acid. At the paratope, typically only certain amino acid changes can be made without materially IPTS / 128584488.1 Page 65 of 180decreasing TNFR1 binding potency. Approaches such as saturation site mutagenesis can be used to determine which paratope positions cannot tolerate substitutions (e.g., without the binding potency of the TNFR1 binding protein materially decreasing) or can tolerate one or more substitutions, as well as which amino acid substitutions are tolerable.

[0125] Sometimes changing amino acids at certain positions, such as the paratope, can alter the function of the synthetic binding protein by increasing or decreasing affinity for the target (i.e., TNFR1). Using SSM, for example, binding affinities can be evaluated by making changes to each identified paratope residue and determining a minimal “threshold” binding affinity to TNFR1 to determine if a paratope residue is tolerant or intolerant to (i) substitution and, if so, (ii) which amino acid(s) can be substituted into the position where a paratope residue is tolerant to substitution. In some embodiments, a threshold binding affinity may be greater than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nM.

[0126] Loss of binding potency can be measured by metrics such as fold-change (e.g., relative to the binding potency prior to one or more substitutions at the paratope), and / or as binding strength (e.g., binding strength that decreases relative to prior to the substitution, as measured in molar values). Binding affinity can be measured using a variety of approaches, including, but not limited to SPR and cell-based assays. One such cell-based approach to measuring binding affinity is a yeast display assay. For example, to evaluate binding affinity in a TNFR1 binding protein, the TNFR1 binding protein can be displayed on the surface of yeast cells, and contacted with a soluble TNFR1 protein. The TNFR1 protein can be presented at varying concentrations, such as 10 pM to 1 ^M. An affinity curve can be generated using concentrations at upper and lower levels of the range, to determine binding affinity of the miniprotein being evaluated. In a given context, e.g., in a particular assay, a change in binding strength (reflected by a numerical increase in binding affinity, e.g., from about 10 nM to about 100 nM is a decrease in strength) beyond a certain threshold can also be considered material, provided however, that binding affinities are all about 250 nM or stronger. It is contemplated that, in some embodiments, a TNFR1 binding protein with a yeast on-cell affinity of weaker than about 250 nM is inefficient at inhibiting TNFR1-mediated signaling in cells. In certain embodiments, a range of acceptable concentrations and / or fold changes within which a substitution is deemed “tolerable” can be determined based on the affinity curves and measurements. That is, in some embodiments, a miniprotein can show binding saturation at 1 nM, but if an amino acid IPTS / 128584488.1 Page 66 of 180substitution decreases the binding affinity to weaker than about 250 nM, the substitution is not considered tolerated, as binding potency to TNFR1 will have decreased materially. In certain embodiments, a binding potency of weaker than about 250 nM (e.g., about 300 nM, about 350 nM, about 400 nM, about 500 nM, or higher, etc.) as measured using on-cell affinity in a yeast display assay, where a TNFR1 binding protein of SEQ ID NO: 997 has an on-cell affinity of 500 pM in a yeast display assay, is considered a material loss of binding potency. Thus, depending upon a given context, a TNFR1 binding protein with an on-cell binding potency of about 250 nM or weaker (e.g., about 300 nM, about 350 nM, about 400 nM, about 500 nM, or higher, etc.) when the comparator has an on-cell binding potency of stronger than about 250 nM (e.g., about 225 nM to about .01 nM or less, e.g., 225 nM, 200 nM, 175 nM, 150 nM, 125 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.01 nM or less ) can be considered a material loss in binding potency. Binding potency can also be measured using other approaches such as SPR. In such embodiments, a material loss in binding strength can be represented by a miniprotein with a binding affinity of about 250 nM or greater, as compared to a TNFR1 binding protein with a binding affinity of less than about 250 nM. In other words, a material loss in binding potency as measured by SPR could occur if a TNFR1 binding protein (e.g., such as provided herein in Tables 1, 2A, 2B, 2C, and 12)having a binding affinity of about 250 nM or stronger (e.g., about 225 nM to about .01 nM or less, e.g., about 225 nM, 200 nM, 175 nM, 150 nM, 125 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.01 nM or less ) is changed to contain one or more substitutions that results in a binding affinity weaker than about 250 nM (e.g., about 300 nM, about 400 nM, about 500 nM, or higher, etc.).

[0127] In certain embodiments, a decrease in binding strength after an amino acid substitution from about 1 nM to above about 250 nM can be considered a material decrease in binding strength. As a result, any amino acid change (e.g., substitution) at a position that results in a binding affinity of about 250 nM or weaker would not be considered tolerable. In some embodiments, when a reference miniprotein (e.g., such as a TNFR1 binding protein as provided herein, e.g., Reference Miniprotein 51, etc.) has an on-cell binding affinity of about 500 pM, a test miniprotein (e.g., having one or more substitutions relative to the reference) with an on-cell binding affinity of about 250 nM or weaker (e.g., about IPTS / 128584488.1 Page 67 of 180275 nM, about 300 nM, about 400 nM, about 500 nM, or higher, etc.) is considered to have a material loss of binding potency relative to the reference miniprotein.

[0128] Tolerated substitutions in TNFR1 binding proteins of the disclosure are those in which a TNFR1 binding protein with one or more substitutions relative to a reference miniprotein (e.g., as provided herein) has a binding affinity of about 250 nM or stronger (e.g., 225, 200, 175, 150, 125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.01 nM or less). Such numbers may also be expressed in fold-change based on the disclosure and parameters provided herein.

[0129] Paratope identity was determined and interrogated across over 900 miniproteins of the disclosure, as discussed in Example 6. A conformational paratope of TNFR1 binding proteins of the disclosure can be set forth according to the formula: X22 - X26 - X31 - X39, where position numbers 22, 26, 31, and 39 correspond to linear, ordinal positions of amino acid residues relative to SEQ ID NO: 993 or 1021, which is a 47-mer. Thus, paratope residues can be found at positions 22, 26, 31, and 39 relative to SEQ ID NO: 993 or 1021. Certain paratope residues can be immutable in that they are completely intolerant to substitution without material loss of function (e.g., as a TNFR1 binding protein, e.g., having a binding potency beyond a threshold, such as provided herein).

[0130] In certain embodiments, the synthetic TNFR1 binding protein binds to TNFR1 through a paratope of the TNFR1 binding protein, which paratope is defined by amino acids X22 - X26 - X31 - X39, wherein X22 is Q, X26 is selected from L, V, I, or A, X31 is selected from E or D, and X39 is selected from Y or F, and wherein the amino acid numbering corresponds to the numbering of SEQ ID NO: 993 or 1021.

[0131] In another aspect, the disclosure provides a means for binding TNFR1. In some embodiments, the means for binding TNFR1 comprises a conformational paratope defined by amino acids X22 - X26 - X31 - X39, wherein X22 is Q, X26 is selected from L, V, I, or A, X31 is selected from E or D, and X39 is selected from Y or F, and wherein the amino acid numbering corresponds to the numbering of SEQ ID NO: 993 or 1021.

[0132] In some embodiments, the conformational paratope is as follows: X22 is Q, X26 is L, X31 is E, X39 is Y. Without material loss of binding potency (e.g., as measured by binding at 11 nM or stronger), X26, in addition to L, can also be V, I, or A, X31, in addition to E, can also be D, and X39, in addition to Y, can also be F. X22 cannot be substituted IPTS / 128584488.1 Page 68 of 180without material loss of binding potency. Other combinations can include those set forth in Table 2C. TABLE 2C. Paratopes of TNFR1 Binding Proteins Amino Acid Position X22 X26 X31 X39 Parato e Number

[0133] In one aspect, the disclosure provides a synthetic TNFR1 binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively; and (ii) an amino acid of SEQ ID NO: 1, wherein D1, D2, and D3 independently comprise any of the following combinations: IPTS / 128584488.1 Page 69 of 180(a) D1 comprises an amino acid sequence of SEQ ID NO: 3, wherein X4 is E or D; X6 is L or I; X7 is E, R, or Q; X8 is Q, R, or E; and X11 is D or E; (b) D2 comprises an amino acid sequence of SEQ ID NO: 4, wherein X20 is Y or A; X21 is Q, R, E, or D; X24 is L, E, I, V, or T; and X28 is D or E; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 5, wherein X33 is P or E; X34 is Q or E; X37 is R or I; X41 is E, D, N, or R; X43 is Y or H; and X45 is R or E.

[0134] Furthermore, L1 can comprise an amino acid sequence of X12GX14IS and / or L2 can comprise an amino acid sequence of X29GED, wherein X12 is Q, E, R, or N; X14 is L, R, or E; and X29 is R, E, or Q.

[0135] In certain embodiments, the synthetic TNFR1 binding protein comprises an amino acid sequence of SEQ ID NO: 1, wherein X4 is E or D; X6 is L or I; X7 is E, R, or Q; X8 is Q, R, or E; X11 is D or E; X12 is Q, E, R, or N; X14 is L, R, or E; X20 is Y or A; X21 is Q, R, E, or D; X24 is L, E, I, V, or T; X28 is D or E; X29 is R, E, or Q; X33 is P or E; X34 is Q or E; X37 is R or I; X41 is E, D, N, or R; X43 is Y or H; and X45 is R or E. In certain embodiments, the TNFR1 binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 6-17.

[0136] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising: an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein, D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively, and wherein D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11 wherein X2 is A or G; X3 is R, A, D, Q, Y, K, H, S, L, N, T, G, or V; X4 is D, A, E, K, S, I, N, V, T, L, Q, F, R, H, Y, or G; X5 is Y, T, G, I, Q, R, A, H, V, S, E, F, K, or D; X7 is E, L, A, R, Q, I, D, K, F, G, V, T, H, N, S, or Y; X8 is R, Y, E, L, V, A, F, Q, N, K, H, T, or G; X10 is R, A, T, V, E, or Y; and X11 is R, D, E, K, S, L, Q, T, F, I, V, G, H, or N; (b) D2 comprises an amino acid sequence of X17X18LX20X21QLX24X25X26X27X28, wherein X17 is D, F, Y, G, H, T, R, A, K, S, H, N, E, V, Q, or I; X18 is V, I, T, Y, F, or D; X20 is E, Y, Q, H, K, N, R, V, T, A, S, F, or L; X21 is Q, Y, D, G, H, I, E, T, V, N, F, R, S, IPTS / 128584488.1 Page 70 of 180or A; X24 is I, S, N, V, G, T, Q, E, D, L, R, F, Y, or A; X25 is D, S, Y, E, Q, or T; X26 is L, I, or V; X27 is L, I, or V; and X28 is N, L, D, R, T, I, Y, A, E, Q, G, V, F, or H; and (c) D3 comprises an amino acid sequence set forth in SEQ ID NO: 21, wherein X33 is E, G, S, A, D, Y, F, Q, I, R, H, K, or N; X34 is N, Q, H, R, Y, V, I, D, A, L, F, K, S, E, or G; X37 is I, V, T, E, R, A, K, or S; X41 is N, Y, L, I, H, D, F, Q, V, S, R, E, A, or T; X43 is Y or F; X44 is I or L; and X45 is E, D, T, R, F, L, S, A, I, Y, V, or N.

[0137] Furthermore, L1 can comprise an amino acid sequence of X12X13X14IX16 and L2 can comprise an amino acid sequence of X29GEX32, wherein X12 is E, Q, S, F, I, K, T, R, L, Y, A, D, N, G, V, or H; X13 is G, R, H, K, S, L, Q, N, I, or Y; X14 is R, Y, G, A, L, I, H, F, V, K, T, E, Q, S, or N; X16 is S, N, G, D, T, or E; X29 is R, I, Q, N, E, S, K, V, L; and X32 is D, F, Y, Q, E, R, N, L, H, A, I, S, V, G, T, or K.

[0138] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein, D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively, and D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of AX3X4X5LX7X8X9RX11, wherein X3 is R, A, D, Q, Y, or K; X4 is D, A, E, K, S, or I; X5 is Y, T, or G; X7 is E, L A, R, Q, I, D, K, or F; X8 is R or Y; X9 is L or Y; and X11 is R, D, E, K, S or L; D2 comprises an amino acid sequence of SEQ ID NO: 24, wherein X17 is D, F, Y, G, or H; X20 is E, Y, Q, H, or K; X21 is Q, Y, D, G, or H; X24 is I, S, N, V, or G; X25 is D, S, or Y; X27 is L or I; and X28 is N, L, D or R; and D3 comprises an amino acid sequence of SEQ ID NO: 25, wherein X33 is E, G, or S; X34 is N, Q, or H; X41 is N, Y, L, or I; X43 is Y or F; and X45 is E, D, T or R; (b) D1 comprises an amino acid sequence of SEQ ID NO: 50, wherein X4 is D or E; and X7 is E or K; D2 comprises an amino acid sequence of SEQ ID NO: 51, wherein X17 is Y, G, or D; and X20 is K, Q, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 52, wherein X33 is E or S; X34 is Q or R; and X41 is N, H, or D; (c) D1 comprises an amino acid sequence of SEQ ID NO: 75, wherein X3 is R, H, S, or K; X4 is D, N, V, T, S, E, L, Q, F, K, or I; X5 is I, Y, Q, or R; X7 is E, L, G, A, K, F, V, IPTS / 128584488.1 Page 71 of 180Q, or R; X8 is R, Y, E, L, V, or A; and X11 is D, K, Q, or T; D2 comprises an amino acid sequence of SEQ ID NO: 76, wherein X17 is D, G, Y, or H; X18 is V, I, or T; X20 is Y, K, R, V, T, E, Q, or A; X21 is Q, G, D, or H; X24 is I, T, or G; X25 is D, E, or Q; and X28 is D, L, T, I, Y, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 77, wherein X33 is A, S, D, E, Y, or F; X34 is Q, Y, V, I, or D; X41 is Y, N, or D; X43 is Y or F; and X45 is E, D, F, or L; (d) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11, wherein X2 is A or G; X3 is R, L, or A; X4 is D, E, K, or R; X5 is A or Y; X7 is E, G, R, L, K, or V; X8 is R, F, Y, or Q; X10 is R or A; and X11 is D or Q; D2 comprises an amino acid sequence of SEQ ID NO: 150, wherein X17 is F, H, T, D, R, A, K, S, or Y; X21 is Q or I; X24 is Q, I, E, or V; X28 is D or N; and D3 comprises an amino acid sequence of SEQ ID NO: 151, wherein X33 is E, G, Y, Q, I, A, F, S, or R; X37 is I or V; X41 is N, F, Y, or L; and X43 is Y or F; (e) D1 comprises an amino acid sequence of SEQ ID NO: 194, wherein X3 is R, D, A, H, N, Q or T; X4 is D, R, N, E, or Q; X7 is E, K, Q, G, R, L, or I; X8 is R, E, A, N, or Q; and X11 is D, F, I, or K; D2 comprises an amino acid sequence of SEQ ID NO: 195, wherein X17 is S, F, Y, H, A, G, R, N, E, D, or T; X18 is V, Y, F, D, or T; X20 is Y, Q, or R; X21 is Q, Y, E, H, or T; X24 is S, I, V, E, or N; X25 is D or E; X26 is L or V; X27 is L or I; and X28 is D, A, or L; and D3 comprises an amino acid sequence of SEQ ID NO: 196, wherein X33 is E, D, F, G, or Y; X34 is Q, I, A, V, N, D, or L; X37 is I or T; X43 is Y or F; and X45 is E, F, or S; (f) D1 comprises an amino acid sequence of AX3X4X5LX7X8LX10X11, wherein X3 is R, Y, A, S, Q, or G; X4 is Q, E, F, D, K, R, or Y; X5 is Y, T, or A; X7 is E, R, G, T, L, H, F, K, A, I, or V; X8 is R, K, or Y; X10 is R or T; and X11 is D or V; D2 comprises an amino acid sequence of SEQ ID NO: 274, wherein X17 is F, D, Y, H, K, S, R, V, A, G, or I; X18 is V or T; X20 is Y, R, E, V, K, H, or T; X21 is Q, Y, V, H, G, or N; X24 is D, I, G, N, or L; X25 is D or E; X26 is L, I, or V; X27 is L or V; and X28 is E, L, D, Q, T, G, or I; and D3 comprises an amino acid sequence of SEQ ID NO: 275, wherein X33 is E, S, D, G, A, H, Y, K, or N; X34 is Q, Y, F, N, R, H, I, K, or Z; X37 is I, E, R, or A; X41 is Y, F, N, L, or I; X43 is Y or F; X44 is I or L; and X45 is E or A; (g) D1 comprises an amino acid sequence of SEQ ID NO: 346, wherein X4 is D, F, or H; and X7 is K, E, G, or R; D2 comprises an amino acid sequence of SEQ ID NO: 347, IPTS / 128584488.1 Page 72 of 180wherein X17 is Y, H, V, or F; X20 is E, K, or Y; X24 is I or S; X26 is L or V; and X27 is L or V; and D3 comprises an amino acid sequence of SEQ ID NO: 348, wherein X34 is F, N, Y, or H; X37 is I or R; X41 is Y or N; and X45 is E, D, or A; (h) D1 comprises an amino acid sequence of SEQ ID NO: 368, wherein X3 is R or H; X4 is D, Y, N, I, or H; X5 is Y or H; X7 is E, A, or K; X8 is R, A, or H; X11 is D, K, or R; D2 comprises an amino acid sequence of SEQ ID NO: 369, wherein X17 is A, G, Q, D, Y, T, N, F, R, K, H, or I; and D3 comprises an amino acid sequence of SEQ ID NO: 370, wherein X34 is H, N, Y, E, G, D, R, F, A, or I; and X41 is L, D, N, Y, or F; (i) D1 comprises an amino acid sequence of SEQ ID NO: 405, wherein X3 is R or Q; X4 is D, Q, V, or S; X5 is Y, T, or V; X7 is E, R, or G; X8 is R or N; and X11 is D, F, or G; D2 comprises an amino acid sequence of SEQ ID NO: 406, wherein X17 is G, Y, A, T, R, F, S, I, Q, or H; X18 is Y or V; X20 is Y, K, E, or T; and X21 is Q or F; and D3 comprises an amino acid sequence of SEQ ID NO: 407, wherein X34 is F, I, D, S, H, R, N, or Y; and X41 is Q, V, Y, N, S, L, I, F, D, R, E, or H; (j) D1 comprises an amino acid sequence of AX3X4X5LX7X8LX10X11, wherein X3 is R, Q, K, A, or E; X4 is D, H, G, S, K, I, L, E, T, or V; X5 is Y, S, E, V, or F; X7 is E, R, G, I, L, or K; X8 is R, T, or G; X10 is R or V; and X11 is D, F, or H; D2 comprises an amino acid sequence of SEQ ID NO: 447, wherein X17 is H, Y, S, G, R, K, A, T, F, or N; X18 is V, D, or I; X20 is Y or Q; X21 is G, Y, Q, H, or F; X24 is I, G, L, V, S, R, or D; X25 is Q, D, or E; X26 is L, I, or V; X27 is L, I, or V; and X28 is D, A, L, R, E, T, V, F, G, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 448, wherein X33 is E, H, N, S, A, or G; X34 is Q, E, R, or V; X37 is I, A, E, or V; X41 is V, Q, F, Y, N, I, L, D, or E; X43 is Y or F; and X45 is E, A, S, L, I, F, or Y; (k) D1 comprises an amino acid sequence of SEQ ID NO: 524, wherein X4 is D, I, N, H, L, K, or A; X5 is K, E, or Y; X8 is R, H, or V; and X11 is D or I; D2 comprises an amino acid sequence of SEQ ID NO: 525, wherein X17 is R, F, N, K, Y, G, H, S, T, or A; X20 is Y, E, N, Q, K, S, or H; X21 is Q, N, E, G, or R; X24 is I or G; and X26 is L or V; and D3 comprises an amino acid sequence of SEQ ID NO: 526, wherein X34 is Q or F; and X41 is Y, F, I, L, D, V, A, E, S, N, or T; (l) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LRX11, wherein X2 is A or G; X3 is R or Q; X4 is D, S, F, L, T, K, or A; X5 is Y or F; X7 is E, G, T, V, or R; X8 is R or L; and X11 is D or V; D2 comprises an amino acid sequence of SEQ ID NO: 565, IPTS / 128584488.1 Page 73 of 180wherein X17 is D or Q; X18 is V or I; X20 is Y, A, E, Q, T, or R; X21 is Q or G; X24 is I, F, G, or Y; and X28 is D, H, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 566, wherein X33 is E, S, G, Y, or D; and X34 is Q or E; X37 is I, A, or V; X41 is F, D, V, Y, I, or L; X43 is Y or F; and X45 is E, Y, I, V, or F; (m) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11, wherein X2 is A or G; X3 is R, Y, S, or A; X4 is E, D, Y, T, A, L, G, F, K, N, or R; X5 is Y, A, I, V, E, G, or S; X7 is E, Q, R, N, A, S, K, or T; X8 is R, G, F, L, Q, K, A, or N; X10 is T, R, V, or E; and X11 is D, L, H, Q, I, N, or F; D2 comprises an amino acid sequence of SEQ ID NO: 605, wherein X17 is H, Q, D, Y, F, T, G, or K; X20 is Y, H, S, K, R, A, E, V, Q, F, L, or T; X21 is Q, G, H, or E; X24 is I, A, or R; X25 is D, T, or Q; and X28 is D, R, I, or F; and D3 comprises an amino acid sequence of SEQ ID NO: 606, wherein X33 is E, S, or G; X34 is L, R, Q, S, or H; X37 is I, A, K, S, or V; X41 is Y, I, L, F, V, D, A, H, or T; X43 is Y or F; X44 is I or L; X45 is E, I, N, Y, or L; (n) D1 comprises an amino acid sequence of SEQ ID NO: 677, wherein X3 is R, Q, V, or G; X4 is D, G, F, R, V, Q, E, L, Y, A, or T; X5 is Y, A, I, V, G, E, or H; X7 is K, E, Y, Q, A, S, L; X8 is R, F, Q, T, K, or Y; and X11 is D, L, R, F, N, Q, K, or V; D2 comprises an amino acid sequence of SEQ ID NO: 678, wherein X17 is F, Q, D, G, or Y; X18 is V, D, or T; X20 is Y, H, Q, R, K, A, T, V, L, or E; and X21 is Q, G, H, or Y; and D3 comprises an amino acid sequence of SEQ ID NO: 679, wherein X34 is K, Y, L, H, F, N, A, R, G, D, E, or I; X37 is I or A; and X41 is V, N, I, F, Y, L, E, or H; (o) D1 comprises an amino acid sequence of SEQ ID NO: 739, wherein X3 is R, V, or H; X4 is D, E, H, G, or K; X5 is Y, D, G, V, or S; X7 is E or R; and X8 is R, F, or V; D2 comprises an amino acid sequence of SEQ ID NO: 740, wherein X18 is V or I; X20 is T, Y, F, K, or V; X21 is Q, R, S, or H; and X25 is Q or D; and D3 comprises an amino acid sequence of SEQ ID NO: 741, wherein X34 is Y, S, G, R, D, F, N, V, A, L, H, I, or K; and X41 is N, F, L, I, Y, V, or D; (p) D1 comprises an amino acid sequence of SEQ ID NO: 787, wherein X4 is D or I; X5 is Y or T; and X8 is R or K; D2 comprises an amino acid sequence of SEQ ID NO: 788, wherein X1 is G, H, D, or Y; X20 is K or Y; and X21 is Q or Y; and D3 comprises an amino acid sequence of SEQ ID NO: 789, wherein X34 is Q, Y, L, N, or G; and X41 is N, F, I, Y, L, V, or D; IPTS / 128584488.1 Page 74 of 180(q) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11, wherein X2 is A or G; X3 is R, H, or Y; X4 is D, Y, F, L, or K; X5 is Y, I, S, A, L, T, or V; X7 is E, Y, I, L, K, S, R, G, A, or Q; X8 is R, L, T, Y, V, or E; X10 is R or Y; and X11 is D, H, or R; D2 comprises an amino acid sequence of SEQ ID NO: 813, wherein X18 is T or V; X20 is Y, Q, K, R, T, S, N, A, or F; and X21 is Q, A, G, H, E, or S; and D3 comprises an amino acid sequence of SEQ ID NO: 814, wherein X41 is V, F, D, Y, I, S, E, or L; (r) D1 comprises an amino acid sequence of SEQ ID NO: 875, wherein X4 is G or D; and X7 is E or A; D2 comprises an amino acid sequence of SEQ ID NO: 876, wherein X17 is H, Y, or G; and X20 is Y, K, or R; and D3 comprises an amino acid sequence of SEQ ID NO: 877, wherein X34 is Q, N, or E; and X41 is N, L, Y, F, D, or I; and (s) D1 comprises an amino acid sequence of SEQ ID NO: 897, wherein X2 is A or G; X3 is R, Y, K, or H; X4 is D, S, L, or G; X5 is Y, T, or K; X7 is R, K, I, E, G, or A; and X11 is D, T, or S; D2 comprises an amino acid sequence of SEQ ID NO: 898, wherein X17 is D, H, Y, F, A, Q, T, R, G, I, E, V, or S; X18 is V, I, or F; X20 is Y, K, T, or S; or X21 is Q, A, Y, or G; and D3 comprises an amino acid sequence of SEQ ID NO: 899, wherein X41 is N, L, D, Y, T, V, K, I, A, E, or F.

[0139] Using the similar labeling of miniproteins (a) through (s), the miniproteins optionally further comprise linker sequences (denoted as L1 and L2) as follows: (a) L1 comprises an amino acid sequence of X12X13X14IX16 and L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E, Q, S, or F; X13 is G or R; X14 is R, Y, G, A, or L; X16 is S, N, G, or D; X29 is R, I, Q, N, E, S, or K; and X32 is D, F, or Y; (b) L1 comprises an amino acid sequence of SEQ ID NO: 990 and L2 comprises an amino acid sequence of X29GEX32, wherein X29 is R, E, or S; and X32 is Q, E, Y, or R; (c) L1 comprises an amino acid sequence of X12X13X14IX16 and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, I, K, T, F, R, S, L, Q, Y, or A; X13 is G, H, K, S, or L; X14 is R, L, I, H, Y, F, A, V, or K; X16 is S, D, or T; and X32 is R, F, Q, Y, N, D, L, H, or A; IPTS / 128584488.1 Page 75 of 180(d) L1 comprises an amino acid sequence of X12X13X14IS and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, D, I, T, R, N, Y, or K; X13 is G or R; X14 is R or K; and X32 is Y, I, R, or L; (e) L1 comprises an amino acid sequence of X12X13X14IS and L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E, N, F, G, S, Y, or V; X13 is G, H, R, K, or Q; X14 is F, R, K, H, I, T, Y, E, or Q; X29 is R, K, or N; and X32 is L, A, R, N, Q, S, Y, F, or V; (f) L1 comprises an amino acid sequence of X12X13X14IX16 and L2 comprises an amino acid sequence of X29GED, wherein X12 is E, F, R, G, Y, L, or H; X13 is G, K, N, H, or R; X14 is R, F, H, Q, or Y; and X29 is R, V, K, or L; (g) L1 comprises an amino acid sequence of X12X13X14IS and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, N, F, D, I, or K; X13 is G, R, or K; X14 is R, E, F, or K; and X32 is D or A; (h) L1 comprises an amino acid sequence of SEQ ID NO: 991 and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X14 is R, A, Y, K, or L; (i) L1 comprises an amino acid sequence of X12X13X14IS and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, T, S, or D; X13 is G, R, or K; and X14 is R, L, or S; (j) L1 comprises an amino acid sequence of X12X13X14IS and L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E, T, H, V, K, N, R, L, or F; X13 is G, N, R, H, or K; X14 is R, V, E, Q, H, K, Y, or F; and X32 is D, Q, or R; (k) L1 comprises an amino acid sequence of X12X13RIS and L2 comprises an amino acid sequence of X29GED, wherein X12 is E, G, S, or K; X13 is G or R; and X29 is R or L; (l) L1 comprises an amino acid sequence of X12X13X14IS and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, L, Q, G, D, Y; X13 is G, H, or I; X14 is Y, F, L, H, V, or I; (m) L1 comprises an amino acid sequence of X12X13X14IX16 and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, K, T, R, I, G, Y, or V; X13 is G, Q, R, K, or H; X14 is R, Y, or K; and X16 is S or E; IPTS / 128584488.1 Page 76 of 180(n) L1 comprises an amino acid sequence of X12X13X14IX16 and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, D, F, T, R, S, N, Y, G, L, Q, or K; X13 is G, K, or Y; X14 is H or R; and X16 is S, T, or D; (o) L1 comprises an amino acid sequence of X12X13X14IX16 and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, Y, S, G, N, I, L; X13 is G, K, or N; X14 is F, Y, A, G, Q, L, I, V, H, K; and X16 is S or D; (p) L1 comprises an amino acid sequence of EX13X14IS and L2 comprises an amino acid sequence of RGEX32, wherein X13 is G, H, or R; X14 is R or Y; and X32 is D, G, S, F, or Y; (q) L1 comprises an amino acid sequence of X12X13X14IX16 and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, L, A, Q, I, H, F, or Y; X13 is G, R, H; X14 is Q, R, F, Y, H, or S; X16 is S, N, or D; and X32 is Y, N, V, F, R, L, S, T, Q, or I; (r) L1 comprises an amino acid sequence of EX13X14IS and L2 comprises an amino acid sequence of RGEX32, wherein X13 is G or K; X14 is R, H, F, or Y; X32 is Y, R, F, or D; and (s) L1 comprises an amino acid sequence of X12X13X14IX16 and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, K, S, or V; X13 is H, G, or R; X14 is R, H, I, Q, V, Y, F, N, or A; X16 is S or D; and X32 is F, N, Y, A, S, L, Q, R, I, E, K.

[0140] In certain aspects, the disclosure provides a synthetic TNFR1 binding protein comprising: an amino acid sequence of SEQ ID NO: 18, wherein X1 is A, S, D, E, F, G, H, I, K, L, N, Q, R, S, V, or Y; X2 is A or G; X3 is R, A, D, Q, Y, K, H, S, L, N, T, G, or V; X4 is D, A, E, K, S, I, N, V, T, L, Q, F, R, H, Y, or G; X5 is Y, T, G, I, Q, R, A, H, V, S, E, F, K, or D; X7 is E, L, A, R, Q, I, D, K, F, G, V, T, H, N, S, or Y; X8 is R, Y, E, L, V, A, F, Q, N, K, H, T, or G; X10 is R, A, T, V, E, or Y; X11 is R, D, E, K, S, L, Q, T, F, I, V, G, H, or N; X12 is E, Q, S, F, I, K, T, R, L, Y, A, D, N, G, V, or H; X13 is G, R, H, K, S, L, Q, N, I, or Y; X14 is R, Y, G, A, L, I, H, F, V, K, T, E, Q, S, or N; X16 is S, N, G, D, T, or E; X17 is D, F, Y, G, H, T, R, A, K, S, H, N, E, V, Q, or I; X18 is V, I, T, Y, F, or D; X20 is E, Y, Q, H, K, N, R, V, T, A, S, F, or L; X21 is Q, Y, D, G, H, I, E, T, V, N, F, R, S, or A; X24 is I, S, N, V, G, T, Q, E, D, L, R, F, Y, or A; X25 is D, S, Y, E, Q, or T; X26 is L, I, or V; X27 is L, I, or V; X28 is N, L, D, R, T, I, Y, A, E, Q, G, V, F, or H; X29 is R, I, Q, IPTS / 128584488.1 Page 77 of 180N, E, S, K, V, or L; X32 is D, F, Y, Q, E, R, N, L, H, A, I, S, V, G, T, or K; X33 is E, G, S, A, D, Y, F, Q, I, R, H, K, or N; X34 is N, Q, H, R, Y, V, I, D, A, L, F, K, S, E, or G; X37 is I, V, T, E, R, A, K, or S; X41 is N, Y, L, I, H, D, F, Q, V, S, R, E, A, or T; X43 is Y or F; X44 is I or L; X45 is E, D, T, R, F, L, S, A, I, Y, V, or N; and X46 is S, G, R, D, T, E, L, I, F, V, or A; X47 is R, V, I, L, T, Y, E, S, K, G, Q, D, N, A, or H.

[0141] In certain embodiments, the disclosure provides a synthetic TNFR1 binding protein comprising the following amino acid sequences: (a) an amino acid sequence of SEQ ID NO: 22, wherein X1 is F, S, R, D, or L; X3 is R, A, D, Q, Y, or K; X4 is D, A, E, K, S, or I; X5 is Y, T, or G; X7 is E, L, A, R, Q, I, D, K, or F; X8 is R or Y; X9 is L or Y; X11 is R, D, E, K, S, or L; X12 is E, Q, S, or F; X13 is G or R; X14 is R, Y, G, A, or L; X16 is S, N, G, or D; X17 is D, F, Y, G, or H; X20 is E, Y, Q, H, or K; X21 is Q, Y, D, G, or H; X24 is I, S, N, V, or G; X25 is D, S, or Y; X27 is L, or I; X28 is N, L, D, or R; X29 is R, I, Q, N, E, S, or K; X32 is D, F, or Y; X33 is E, G, or S; X34 is N, Q, or H; X41 is N, Y, L, or I; X43 is Y or F; X45 is E, D, T, or R; X46 is S, G, or R; and X47 is R, V, I, L, T, Y, E, or S; (b) an amino acid sequence of SEQ ID NO: 49, wherein X1 is S, F, V, or R; X4 is D or E; X7 is E or K; X17 is Y, G, D; X20 is K, Q, or N; X29 is R, E, or S; X32 is Q, E, Y, or R; X33 is E or S; X34 is Q or R; X41 is N, H, or D; X46 is S, D, or R; and X47 is K, R, E, L, G, Q, or I; (c) an amino acid sequence of SEQ ID NO: 74, wherein X1 is L, S, Y, F, G, T, I, V, K, H, D, Q, or R; X3 is R, H, S, or K; X4 is D, N, V, T, S, E, L, Q, F, K, or I; X5 is I, Y, Q, or R; X7 is E, L, G, A, K, F, V, Q, or R; X8 is R, Y, E, L, V, or A; X11 is D, K, Q, or T; X12 is E, I, K, T, F, R, S, L, Q, Y, or A; X13 is G, H, K, S, or L; X14 is R, L, I, H, Y, F, A, V, or K; X16 is S, D, or T; X17 is D, G, Y, H; X18 is V, I, or T; X20 is Y, K, R, V, T, E, Q, or A; X21 is Q, G, D, or H; X24 is I, T, or G; X25 is D, E, or Q; X28 is D, L, T, I, Y, or N; X32 is R, F, Q, Y, N, D, L, H, or A; X33 is A, S, D, E, Y, or F; X34 is Q, Y, V, I, D; X41 is Y, N, or D; X43 is Y or F; X45 is E, D, F, or L; X46 is S, D, T, E, or L; and X47 is R, T, L, Q, I, D, S, Y, or N; (d) an amino acid sequence of SEQ ID NO: 148, wherein X1 is S or V; X2 is A or G; X3 is R, L, or A; X4 D, E, K, or R; X5 is A or Y; X7 is E, G, R, L, K, or V; X8 is R, F, Y, or Q; X10 is R or A; X11 is D or Q; X12 is E, D, I, T, R, N, Y, or K; X13 is G or R; X14 is R or K; X17 is F, H, T, D, R, A, K, S, or Y; X21 is Q or I; X24 is Q, I, E, or V; IPTS / 128584488.1 Page 78 of 180X28 is D or N; X32 is Y, I, R, or L; X33 is E, G, Y, Q, I, A, F, S, or R; X37 I or V; X41 is N, F, Y, or L; X43 is Y or F; X46 is S or G; X47 is R, T, E, Q, L, N, Y, I, or F; (e) an amino acid sequence of SEQ ID NO: 193, wherein X1 is S, V, Y, F, K, R, G, or A; X3 is R, D, A, H, N, Q, or T; X4 is D, R, N, E, or Q; X7 is E, K, Q, G, R, L, or I; X8 is R, E, A, N, or Q; X11 is D, F, I, or K; X12 is E, N, F, G, S, Y, or V; X13 is G, H, R, K, or Q; X14 is F, R, K, H, I, T, Y, E, or Q; X17 is S, F, Y, H, A, G, R, N, E, D, or T; X18 is V, Y, F, D, or T; X20 is Y, Q, or R; X21 is Q, Y, E, H, or T; X24 is S, I, V, E, or N; X25 is D or E; X26 is L or V; X27 is L or I; X28 is D, A, or L; X29 is R, K, or N; X32 is L, A, R, N, Q, S, Y, F, or V; X33 is E, D, F, G, or Y; X34 Q, I, A, V, N, D, or L; X37 is I or T; X43 is Y or F; X45 is E, F, or S; X46 is S, I, E, F, V, A, or D; and X47 is R, A, E, T, L, I, N, G, Q, or S; (f) an amino acid sequence of SEQ ID NO: 272, wherein X1 is S, R, K, F, or L; X3 is R, Y, A, S, Q, or G; X4 is Q, E, F, D, K, R, or Y; X5 is Y, T, or A; X7 is E, R, G, T, L, H, F, K, A, I, or V; X8 is R, K, or Y; X10 is R or T; X11 is D or V; X12 is E, F, R, G, Y, L, or H; X13 is G, K, N, H, or R; X14 is R, F, H, Q, or Y; X16 is S or D; X17 F, D, Y, H, K, S, R, V, A, G, or I; X18 is V or T; X20 is Y, R, E, V, K, H, or T; X21 is Q, Y, V, H, G, or N; X24 is D, I, G, N, or L; X25 is D or E; X26 is L, I, or V; X27 is L or V; X28 is E, L, D, Q, T, G, or I; X29 is R, V, K, or L; X33 is E, S, D, G, A, H, Y, K, or N; X34 is Q, Y, F, N, R, H, I, K, or A; X37 is I, E, R, or A; X41 is Y, F, N, L, or I; X43 is Y or F; X44 is I or L; X45 is E or A; X46 is S, L, D, I, E, or A; X47 is R, V, L, T, I, G, Q, E, F, S, or Y; (g) an amino acid sequence of SEQ ID NO: 345, wherein X4 is D, F, or H; X7 is K, E, G, or R; X12 is E, N, F, D, I, or K; X13 is G, R, or K; X14 is R, E, F, or K; X17 is Y, H, V, or F; X20 is E, K, or Y; X24 is I or S; X26 is L or V; X27 is L or V; X32 is D or A; X34 is F, N, Y, or H; X37 is I or R; X41 is Y or N; X45 is E, D, or A; X46 is S, L, or F; X47 is R, L, V, Q, E, N, or I; (h) an amino acid sequence of SEQ ID NO: 367, wherein X1 is S, F, or V; X3 is R or H; X4 is D, Y, N, I, or H; X5 is Y or H; X7 is E, A, or K; X8 is R, A, or H; X11 is D, K, or R; X14 is R, A, Y, K, or L; X17 is A, G, Q, D, Y, T, N, F, R, K, H, or I; X18 is V, D, F, or T; X20 is H, K, V, or Y; X21 is Q, H, or Y; X34 is H, N, Y, E, G, D, R, F, A, or I; X41 is L, D, N, Y, or F; and X47 is R or I; IPTS / 128584488.1 Page 79 of 180(i) an amino acid sequence of SEQ ID NO: 404, wherein X1 is S, I, or Y; X3 is R or Q; X4 is D, Q, V, is S; X5 is Y, T, or V; X7 is E, R, or G; X8 is R or N; X11 is D, F, or G; X12 is E, T, S, or D; X13 is G, R, or K; X14 is R, L, or S; X17 is G, Y, A, T, R, F, S, I, Q, or H; X18 is Y or V; X20 is Y, K, E, or T; X21 is Q or F; X34 is F, I, D, S, H, R, N, or Y; X41 is Q, V, Y, N, S, L, I, F, D, R, E, or H; (j) an amino acid sequence of SEQ ID NO: 445, wherein X1 is S, Q, F, R, or K; X3 is R, Q, K, A, or E; X4 is D, H, G, S, K, I, L, E, T, V; X5 is Y, S, E, V, or F; X7 is E, R, G, I, L, or K; X8 is R, T, or G; X10 is R or V; X11 is D, F, or H; X12 is E, T, H, V, K, N, R, L, or F; X13 is G, N, R, H, or K; X14 is R, V, E, Q, H, K, Y, or F; X17 is H, Y, S, G, R, K, A, T, F, or N; X18 is V, D, or I; X20 is Y or Q; X21 is G, Y, Q, H, or F; X24 is I, G, L, V, S, R, or D; X25 is Q, D, or E; X26 is L, I, or V; X27 is L, I, or V; X28 is D, A, L, R, E, T, V, F, G, or N; X29 is R or K; X32 is D, Q, or R; X33 is E, H, N, S, A, or G; X34 is Q, E, R, or V; X37 is I, A, E, or V; X41 is V, Q, F, Y, N, I, L, D, or E; X43 is Y or F; X45 is E, A, S, L, I, F, or Y; X46 is S, L, or E; and X47 is R or Y; (k) an amino acid sequence of SEQ ID NO: 523, wherein X1 is S or K; X4 is D, I, N, H, L, K, or A; X5 is K, E, or Y; X8 is R, H, or V; X11 is D or I; X12 is E, G, S, or K; X13 is G or R; X17 is R, F, N, K, Y, G, H, S, T, or A; X20 is Y, E, N, Q, K, S, or H; X21 is Q, N, E, G, or R; X24 is I or G; X26 is L or V; X29 is R or L; X34 is Q or F; and X41 is Y, F, I, L, D, V, A, E, S, N, or T; (l) an amino acid sequence of SEQ ID NO: 563, wherein X1 is V, S, or L; X2 is A or G; X3 is R or Q; X4 is D, S, F, L, T, K, or A; X5 is Y or F; X7 is E, G, T, V, or R; X8 is R or L; X11 is D or V; X12 is E, L, Q, G, D, or Y; X13 is G, H, or I; X14 is Y, F, L, H, V, or I; X17 is D or Q; X18 is V or I; X20 is Y, A, E, Q, T, or R; X21 is Q or G; X24 is I, F, G, or Y; X28 is D, H, or N; X33 is E, S, G, Y, or D; X34 is Q or E; X37 is I, A, or V; X41 is F, D, V, Y, I, or L; X43 is Y or F; X45 is E, Y, I, V, or F; X46 is S, D, E, L, or A; and X47 is S, V, R, E, or L; (m) an amino acid sequence of SEQ ID NO: 603, wherein X1 is S, R, K, I, Q, L, Y, or F; X2 is A or G; X3 is R, Y, S, or A; X4 is E, D, Y, T, A, L, G, F, K, N, or R; X5 is Y, A, I, V, E, G, or S; X7 is E, Q, R, N, A, S, K, or T; X8 is R, G, F, L, Q, K, A, or N; X10 is T, R, V, or E; X11 is D, L, H, Q, I, N, or F; X12 is E, K, T, R, I, G, Y, or V; X13 is G, Q, R, K, or H; X14 is R, Y, or K; X16 is S or E; X17 is H, Q, D, Y, F, T, G, or K; X20 is Y, H, S, K, R, A, E, V, Q, F, L, or T; X21 is Q, G, H, or E; X24 is I, A, or R; IPTS / 128584488.1 Page 80 of 180X25 is D, T, or Q; X28 is D, R, I, or F; X33 is E, S, or G; X34 is L, R, Q, S, or H; X37 is I, A, K, S, or V; X41 is Y, I, L, F, V, D, A, H, or T; X43 is Y or F; X44 is I or L; X45 is E, I, N, Y, or L; X46 is S, E, D, or L; and X47 is R, H, E, I, A, V, L, S, T, Q, or K; (n) an amino acid sequence of SEQ ID NO: 676, wherein X1 is S, G, K, V, Q, or E; X3 is R, Q, V, or G; X4 is D, G, F, R, V, Q, E, L, Y, A, or T; X5 is Y, A, I, V, G, E, or H; X7 is K, E, Y, Q, A, S, or L; X8 is R, F, Q, T, K, or Y; X11 is D, L, R, F, N, Q, K, or V; X12 is E, D, F, T, R, S, N, Y, G, L, Q, or K; X13 is G, K, or Y; X14 is H or R; X16 is S, T, or D; X17 is F, Q, D, G, or Y; X18 is V, D, or T; X20 is Y, H, Q, R, K, A, T, V, L, or E; X21 is Q, G, H, or Y; X34 is K, Y, L, H, F, N, A, R, G, D, E, or I; X37 is I or A; and X41 is V, N, I, F, Y, L, E, or H; (o) an amino acid sequence of SEQ ID NO: 738, wherein X1 is S, F, Q, R, or I; X3 is R, V, or H; X4 is D, E, H, G, or K; X5 is Y, D, G, V, or S; X7 is E or R; X8 is R, F, or V; X12 is E, Y, S, G, N, I, or L; X13 G, K, or N; X14 is F, Y, A, G, Q, L, I, V, H, or K; X16 is S or D; X18 is V or I; X20 is T, Y, F, K, or V; X21 is Q, R, S, or H; X25 is Q or D; X34 is Y, S, G, R, D, F, N, V, A, L, H, I, or K; X41 is N, F, L, I, Y, V, or D; and X47 is R or E; (p) an amino acid sequence of SEQ ID NO: 786, wherein X1 is S, K, or Y; X4 is D or I; X5 is Y or T; X8 is R or K; X13 is G, H, or R; X14 is R or Y; X17 is G, H, D, or Y; X20 is K or Y; X21 is Q or Y; X32 is D, G, S, F, or Y; X34 is Q, Y, L, N, or G; X41 is N, F, I, Y, L, V, or D; and X47 is R, I, or T; (q) an amino acid sequence of SEQ ID NO: 811, wherein X1 is S, H, G, L, N, Y, Q, or K; X2 is A or G; X3 is R, H, or Y; X4 is D, Y, F, L, or K; X5 is Y, I, S, A, L, T, or V; X7 is E, Y, I, L, K, S, R, G, A, or Q; X8 is R, L, T, Y, V, or E; X10 is R or Y; X11 is D, H, or R; X12 is E, L, A, Q, I, H, F, or Y; X13 is G, R, or H; X14 is Q, R, F, Y, H, or S; X16 is S, N, or D; X18 is T or V; X20 is Y, Q, K, R, T, S, N, A, or F; X21 is Q, A, G, H, E, or S; X32 is Y, N, V, F, R, L, S, T, Q, or I; and X41 V, F, D, Y, I, S, E, or L; (r) an amino acid sequence of SEQ ID NO: 874, wherein X1 is S or F; X4 is G or D; X7 is E or A; X13 is G or K; X14 is R, H, F, or Y; X17 is H, Y, or G; X20 is Y, K, or R; X32 is Y, R, F, or D; X34 is Q, N, or E; X41 is N, L, Y, F, D, or I; and X47 is R, E; and (s) an amino acid sequence of SEQ ID NO: 896, wherein X1 is S, F, R, Q, G, V; X2 is A or G; X3 is R, Y, K, or H; X4 is D, S, L, or G; X5 is Y, T, or K; X7 is R, K, I, E, G, or A; X11 is D, T, or S; X12 is E, K, S, or V; X13 is H, G, or R; X14 is R, H, I, Q, V, IPTS / 128584488.1 Page 81 of 180Y, F, N, or A; X16 is S or D; X17 is D, H, Y, F, A, Q, T, R, G, I, E, V, or S; X18 is V, I, or F; X20 is Y, K, T, or S; X21 is Q, A, Y, or G; X32 is F, N, Y, A, S, L, Q, R, I, E, or K; X41 is N, L, D, Y, T, V, K, I, A, E, or F; and X47 is R or E.

[0142] An exemplary synthetic TNFR1 binding protein can comprise an amino acid sequence selected from any of SEQ ID NOs: 26-48, 53-73, 78-147, 152-192, 197-271, 276- 344, 349-366, 371-403, 408-444, 449-522, 527-562, 567-602, 607-675, 680-737, 742-785, 790-810, 815-873, 878-895, and 900-957.

[0143] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively, and D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of SEQ ID NO: 959, wherein X5 is Y or T; X6 is L or I; X7 is R, Q, E, D, or K; X8 is R or Y; X9 is L or Y; and X11 is D or E; (b) D2 comprises an amino acid sequence of SEQ ID NO: 960, wherein X17 is D, G, or H; X20 is E, K, or Y; X21 is G or Q; X23 is L or Y; and X24 is N or I; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 961, wherein X33 is E or S; and X43 is S or Y.

[0144] It is contemplated that such a miniprotein optionally further comprises linker sequences (L1 and L2) wherein (i) L1 comprises an amino acid sequence of X12GX14IS; and (ii) L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E or Q; X14 is Y or R; X29 is R, I, E, Q, N, or S; and X32 is D or Y.

[0145] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising an amino acid sequence of SEQ ID NO: 958, wherein X5 is Y or T; X6 is L or I; X7 is R, Q, E, D, or K; X8 is R or Y; X9 is L or Y; X11 is D or E; X12 is E or Q; X14 is Y or R; X17 is D, G, or H; X20 is E, K, or Y; X21 G or Q; X24 is N or I; X29 is R, I, E, Q, N, or S; X32 is D or Y; X33 is E or S; X43 is F or Y; and X47 is R, Y, E, or L.

[0146] Exemplary synthetic TNFR1 binding proteins can comprise an amino acid sequence selected from any of SEQ ID NOs: 878 and 962-982. In certain embodiments, the TNFR1 binding protein comprising an amino acid sequence of SEQ ID NO: 878. IPTS / 128584488.1 Page 82 of 180

[0147] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively, and (ii) an amino acid sequence of SEQ ID NO: 993, wherein D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of SEQ ID NO: 994, wherein X4 is D, Q, or N; X5 is Y or F; X7 is E, R, H, A, S, or K; X8 is R, Y, L, or V; and X11 is D, K, Q, R, N, S, I, or T; (b) D2 comprises an amino acid sequence of SEQ ID NO: 995, wherein X18 is V, T, N, or I; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 996, wherein X34 is Q, D, E, or N; X40 is A or V; and X41 is L or D.

[0148] Furthermore, L1 can comprise an amino acid sequence of X12GX14IS and L2 comprises an amino acid sequence of X29GED, wherein X12 is E or A; X14 is Y or L; and X29 is R, I, or L.

[0149] In certain embodiments, the synthetic TNFR1 binding protein comprises an amino acid sequence of SEQ ID NO: 993, wherein X1 is S, H, R, Y, V, F, or L; X4 is D, Q, or N; X5 is Y or F; X7 is E, R, H, A, S, or K; X8 is R, Y, L, or V; X11 is D, K, Q, R, N, S, I, or T; X12 is E or A, or N; X14 is Y or L; X18 is V, T, N, or I; X29 is R, I, or L; X34 is Q, D, E, or N; X40 is A or V; X41 is L or D; and X47 is R, E, L, Q, or T.

[0150] In certain embodiments, the TNFR1 binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 997-1020.

[0151] In another aspect, the disclosure provides a synthetic TNFR1 binding protein comprising an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively, and (ii) an amino acid sequence of SEQ ID NO: 1021, wherein D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of SEQ ID NO: 1022, wherein X8 is R or Y; and X11 is D, K, or R; IPTS / 128584488.1 Page 83 of 180(b) D2 comprises an amino acid sequence of SEQ ID NO: 1023; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 1024, wherein X41 is L or D.

[0152] Furthermore, L1 can comprise an amino acid sequence of X12GYIS and L2 comprises an amino acid sequence of X29GED, wherein X12 is E or A; and X29 is R or I.

[0153] In certain embodiments, the synthetic TNFR1 binding protein comprises an amino acid sequence of SEQ ID NO: 1021, wherein X8 R or Y; X11 is D, K, or R; X12 is E or A; X29 is R or I; X41 is L or D; and X47 is R or E.

[0154] In certain embodiments, the TNFR1 binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 997, 998, or 1020.

[0155] The synthetic TNFR1 binding proteins disclosed herein can have a the binding affinity between about 10 ^M to about 0.1 n ^; about 7.5 ^M to about 0.75 nM; about 5 ^M to about 0.5 nM ^ about 2.5 ^M to about 0.25 nM; about 1 ^M to about 1 n ^; about 0.5 ^M to about 1 nM; about 0.25 ^M to about 1 n ^ ^ ^about 0.10 ^M to about 1 n ^ ^ ^about 75 n ^ to about 1 n ^ ^ ^about 50 n ^ to about 1 n ^ ^ ^about 25 n ^ to about 1 n ^ ^ ^about 10 n ^ to about 1 n ^ ^ ^and about 5 n ^ to about 1 n ^. Furthermore, the synthetic TNFR1 binding protein can have a binding affinity stronger than about 10 ^M, about 7.5 ^M, about 5 ^M, about 2.5 ^M, about 1 ^M, about 0.75 ^M, about 0.5 ^M, about 0.25 ^M, about 0.1 ^M, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, and about 0.1 nM.

[0156] In some embodiments, a threshold binding affinity may be greater than about 10 ^M, about 1 ^M, about 100 nM, about 10 nM, or about 1 nM.

[0157] It is contemplated that optimization of synthetic binding proteins may be achieved using optimized designs, such as, for example, modifications of one or more amino acids by substitution at one or more positions with a different amino acid. Optimization, such as by amino acid modifications, allows tunability of certain characteristics such as changes to (e.g., increases in) binding affinity and / or avidity.

[0158] Synthetic TNFR1 binding proteins can be optimized by affinity maturation techniques. For example, affinity maturation may be used on a sequence of a binding protein to create another synthetic TNFR1 binding protein with at least the same or better IPTS / 128584488.1 Page 84 of 180selectivity and / or affinity for TNFR1 as compared to the starting sequence. Affinity maturation can be accomplished using techniques known to those of ordinary skill in the art, including, for example, generating libraries using error prone PCR, degenerate codons, synthetic oligonucleotide pools, or a combination thereof. These libraries can than be transformed into yeast and improved variants may be isolated by methods such as magnetic, flow cytometric, and / or FACS-based approaches. Computational design / redesign strategies may also be used when affinity maturing proteins and computer programs for implementing such approaches are known in the art. Prior to affinity maturation, synthetic binding proteins may be characterized to determine functional and structural features, such as binding affinity (e.g., for TNFR1) and conformation.

[0159] Synthetic TNFR1 binding proteins provided herein are engineered to have certain characteristics (e.g., binding affinity / avidity, e.g., for a target, e.g., for TNFR1). Various in silico, in vitro, and in vivo characterization assays may be used to evaluate these TNFR1 binding proteins. For example, binding assays can be used to determine binding specificity to a target, e.g., TNFR1, as compared to binding to another receptor, e.g., TNFR2. Other assays can be used to determine binding affinity of a binding protein, e.g., a TNFR1 binding protein for its target, which can include for example, surface plasmon resonance (SPR), and flow cytometry.

[0160] The synthetic TNFR1 binding proteins of the present disclosure are designed to have certain stability characteristics. For example, a binding protein is stable in that it may retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to TNFR1 upon cooling to room temperature after thermal denaturation at 95oC in a solution (e.g., phosphate buffered saline (PBS)) for at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more minutes relative to the synthetic TNFR1 binding protein prior to thermal denaturation.

[0161] In some embodiments, a synthetic TNFR1 binding protein is stable in that it may retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to TNFR1 after incubation at 37oC (e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more hours relative to the synthetic TNFR1 binding protein prior to incubation.

[0162] Synthetic TNFR1 binding proteins of the present disclosure may also display stability in resistance to chemical denaturation and / or retention of stability after exposure to IPTS / 128584488.1 Page 85 of 180chemical denaturants. For example, a synthetic TNFR1 binding protein may be stable in that it may retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to TNFR1 in PBS following exposure to a denaturing chemical (e.g., 4M urea) at room temperature for about 15 minutes, 30 minutes, 45 minutes, 1, 2, 3, 4, 5, 6, 7, 8 or more hours relative to the binding affinity of the synthetic TNFR1 binding protein prior to exposure to the chemical denaturant.

[0163] Synthetic TNFR1 binding proteins engineered, developed, and produced herein are selected and / or specific for TNFR1. That is, in some embodiments, a synthetic TNFR1 binding proteins does not bind to TNFR2. In some embodiments, a synthetic TNFR1 binding protein binds to TNFR2, but binds to TNFR1 with a much greater affinity. For example, the binding affinity of a synthetic TNFR1 binding protein may be between 1 and 1000- fold greater than that for TNFR2.

[0164] Affinity of a synthetic TNFR1 binding protein may be modified and may vary depending on modifications made to, for example, its primary sequence. A binding affinity may be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70- fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, at least 250-fold greater, at least 500-fold greater, or at least 1000-fold greater than the affinity of the synthetic TNFR1 binding protein for an unrelated target (e.g., TNFR2). IV. SYNTHESIS OF TNFR1 BINDING PROTEINS

[0165] The synthetic TNFR1 binding proteins described herein may be produced by methods known to those of ordinary skill in the art. Methods may include, for example, biological approaches, such as recombinant approaches and / or chemical approaches, such as solid phase and / or liquid phase chemical synthesis, etc., or combinations thereof.

[0166] With regard to recombinant approaches, a variety of methodologies can be implemented to produce the binding proteins disclosed herein. For example, DNA molecules encoding the binding proteins can be synthesized chemically and / or cloned / produced using recombinant DNA methodologies. The resulting DNA molecules encoding binding proteins of interest can be ligated to other nucleotide sequences, IPTS / 128584488.1 Page 86 of 180including, for example, expression control sequences, to produce a gene expression construct (i.e., expression vector). Thereafter, the resulting expression vectors are introduced into host cells using conventional transfection or transformation techniques. Exemplary host cells include E. coli cells, Pichia Pastoris cells, Saccharomyces cerevisiae cells, Kluyveromyces lactis cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells (e.g., Hep G2). The transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the binding proteins.

[0167] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., T7, lac, Trp or Tac, and, in some contexts, a prokaryotic signal sequence or fusion to a protein such as, e.g., Trx, MBP, SUMO, or OsmY. The expressed protein may be secreted. The expressed protein can be harvested after disruption of the cells by French press or sonication (e.g., in the presence of 4 – 6M urea). Alternatively, or in addition, the binding proteins can be harvested and purified or isolated from cell extracts using techniques known in the art, e.g., affinity tags such as glutathione- S-transferase (GST) or histidine tags. Protease cleavage with SUMO Protease (Ulp), thrombin, enterokinase, TEV protease, 3C protease may be used to cleave affinity tags and fusion proteins from the miniprotein binder. The protein may be further purified with reverse phase HPLC using a C-18 column and eluted in a solvent gradient (e.g., gradient of acetonitrile). The protein may then be lyophilized to remove solvent and may be resuspended in phosphate buffered saline. Purification by reverse phase HPLC may be used to remove endotoxin from samples expressed in E. coli.

[0168] If the engineered gene is expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. Optionally, the vector or gene construct may contain enhancers. The vector may also optionally contain fusion domains which can be used to facilitate expression and secretion. Vectors may also optionally contain enzyme (e.g., protease) cleavage sites. The gene construct can be introduced into eukaryotic host cells using conventional transfection (e.g., for mammalian) and transformation (e.g., for yeast). IPTS / 128584488.1 Page 87 of 180

[0169] In addition, the synthetic binding proteins may be produced in cell-free systems. For example, chemical synthesis such as organic chemical synthesis using liquid and / or solid phase chemical processes may be used. Such processes and tools for performing such processes, such as various automatic synthesizers, are well known to those of ordinary skill in the art and such tools are widely commercially available. More specifically, methods of chemically synthesizing polypeptides are well known in the art and include, but are not limited to, solid-phase peptide synthesis, liquid-phase peptide synthesis, and organic synthesis methods. In some synthetic approaches, an amino group of one amino acid (or amino acid derivative) is linked to a carboxyl group of another amino acid (or amino acid derivative) that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide (DCC). When the free amino group attacks the activated carboxyl group, a peptide bond is formed and dicyclohexylurea is released. In such methods, other potentially reactive groups (such as the α-amino group of the N-terminal amino acid or amino acid derivative and the carboxyl group of the C-terminal amino acid or amino acid derivative) may be blocked (“protected”) from participating in the chemical reaction. Thus, only particular active groups react such that the desired product is formed. Blocking groups useful for this purpose include, without limitation, tertbutoxycarbonyl groups (t-Boc) and benzoyloxycarbonyl groups to protect amine groups; and simple esters (such as methyl and ethyl groups) and benzyl esters to protect carboxyl groups. Blocking groups can typically be subsequently removed with a treatment that leaves peptide bonds intact (for example, treatment with dilute acid). This process of protecting reacting groups that should not react, coupling to form a peptide bond, and deprotecting reactive groups may be repeated. A peptide may be synthesized by sequentially adding amino acids to a growing peptide chain.

[0170] Both liquid-phase and solid phase peptide synthesis methods can be used to make the binding proteins described herein. In solid-phase peptide synthesis, the growing peptide chain is typically linked to an insoluble matrix (such as, for example, polystyrene beads) by linking the carboxyterminal amino acid to the matrix. At the end of synthesis, the peptide can be released from the matrix using a cleaving reagent that does not disrupt peptide bonds, such as hydrofluoric acid (HF). Protecting groups are also typically removed at this time. Automated, high throughput, and / or parallel peptide synthesis methods may also be used in accordance with the disclosure. For more information about peptide synthesis methods, see, e.g., Merrifield (1969) ADV. ENZYMOL. RELAT. AREAS IPTS / 128584488.1 Page 88 of 180MOL. BIOL., 32: 221-96; Fridkin et al. (1974) ANN. REV. BIOCHEM. 43(0): 419-43; Merrifield (1997) METH. ENZYMOL. 289: 3-13; Sabatino et al. (2009) CURR. OPIN. DRUG DISCOV. DEVEL., 11(6): 762-70.

[0171] Once synthesized, the binding proteins can be purified using standard approaches including, for example, chromatographic (e.g., reverse phase HPLC) and affinity binding approaches. The resulting binding proteins can then be characterized using a variety of chemical, biological and biophysical approaches. V. CHARACTERIZATION OF TNFR1 BINDING PROTEINS A. Biophysical Characterization

[0172] The synthetic binding proteins described herein may be characterized using a variety of approaches to determine, e.g., secondary and tertiary conformation, binding affinity, binding selectivity, stability (e.g., thermostability, chemical stability, propensity to degrade, etc.), solubility, etc.

[0173] For example, protein conformation may be measured via circular dichroism spectroscopy, infrared spectroscopy, NMR, X-ray crystallography, cryo-electron microscopy and AlphaFold (alphafold.ebi.ac.uk / ). Binding affinity and / or selectivity may be determined using assays such as flow cytometric analyses using, e.g., yeast or mammalian cells, biolayer interferometry and / or surface plasmon resonance measurements, each of which will be able to determine different types and specificities of binding.

[0174] Binding affinity and / or avidity can be determined by measuring the equilibrium dissociation constant (KD) of a synthetic TNFR1 binding protein to a target. In some embodiments, the binding affinity (KD) of synthetic TNFR1 binding proteins in the range of 10-5M or less, or ranging down to 10-16M or lower, (e.g., about 10-6,10-7, 10-8, 10-9, 10-10, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16M or less).

[0175] In some embodiments, the synthetic TNFR1 binding protein comprises a binding affinity characterized by a dissociation constant ranging from about 100 pM to 10 ^M. In some embodiments, the binding affinity is between about 0.1 nM to about 10 ^M; about 0.75 nM to about 7.5 ^M; about 0.5 nM to about 5 ^M; about 0.25 nM to about 2.5 ^M; about 1 nM to about 1 ^M; about 1 nM to about 0.5 ^M; about 1 nM to about 0.25 ^M; about 1 nM to about 0.10 ^M; about 1 nM to about 75 nM; about 1 nM to about 50 nM; about 1 nM to about 25 nM; about 1 nM to about 10 nM; and about 1 nM to about 5 nM. IPTS / 128584488.1 Page 89 of 180In some embodiment, the binding affinity is stronger than about 10 ^M, about 7.5 ^M, about 5 ^M, about 2.5 ^M, about 1 ^M, about 0.75 ^M, about 0.5 ^M, about 0.25 ^M, about 0.1 ^M, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, and about 0.1 nM. One of ordinary skill in the art will readily know how to calculate equilibrium dissociation constants using measured Ka (1 / sec) and Kd (1 / secM) of the synthetic TNFR1 binding proteins.

[0176] Other analytical techniques (some of which are also used for synthesis and purification) include, without limitation, HPLC, LCMS, quantitative thin layer chromatography and others known to those of skill in the art. Stability can be measured using assays that expose binding proteins to elevated temperatures (e.g., 37 °C, e.g., 95 °C, etc.) and / or chemical denaturants (e.g., urea, and guanidine hydrochloride) and then observe whether the protein refolds into its pre-exposure structure / conformation and / or regains binding activity to a given target molecule. Degradation can be evaluated using techniques such as reverse phase HPLC or gel electrophoresis to monitor resistance of a synthetic binding protein to degradation. B. Biochemical Characterization

[0177] The biological activity of the binding proteins can be determined via in vitro and in vivo assays (see, e.g., Examples 7 and 8). Such assays can be used to determine whether a binding protein has agonistic or antagonistic properties. For example, the suitable assays can be performed to determine whether a synthetic TNFR1 binding protein disclosed herein can, e.g., partially or completely inhibit downstream TNFR1-mediated signaling.

[0178] Various assays may be used to evaluate efficacy of synthetic TNFR1 binding proteins to determine their ability to inhibit TNFR1-mediated signaling. Certain assays can measure the interference of TNFR1 signaling by its natural ligands (e.g., TNF ^, e.g., lymphotoxin ^). In the assay, a synthetic TNFR1 binding protein interferes with the binding of a TNFR1 ligand (e.g., a soluble TNF ^ homotrimer) to TNFR1 and its downstream signaling activity.

[0179] Assays conducted with cells, such as mouse (e.g., L929) or human (e.g., HEK293) cells, may be used to evaluate and characterize synthetic TNFR1 binding proteins. For example, the cells may have a visualizable reporter that is detectable upon TNFR1- mediated signaling. In some embodiments, TNFR1 signaling is measured by contacting a IPTS / 128584488.1 Page 90 of 180population of cells with a TNFR1 ligand and measuring cell death (e.g., TNFR1-mediated cell death). The amount of cell death can be reduced or eliminated by contacting the population of cells (before, concomitant with, or after exposure to a ligand) with a synthetic TNFR1 binding protein.

[0180] Characterization assays may also be conducted in vivo. For example, synthetic TNFR1 binding proteins may be tested for efficacy in interference with TNFR1-mediated signaling by comparing survival of mice treated with a normally lethal dose of a TNFR1 ligand such as TNF ^ in combination with a synthetic TNFR1 binding protein as compared to survival in mice treated with a TNFR1 ligand and a control protein such as TNFR1 ligand antibody or a control binding protein that does not bind TNFR1. In certain embodiments, synthetic TNFR1 binding proteins have at least about the same ability as a TNFR1 ligand antibody (e.g., a TNF ^ antibody) to reduce or prevent death, and, in some instances, synthetic TNFR1 binding proteins result in greater survival than a TNFR1 ligand antibody. VI. TNFR1-BINDING PROTEIN CONJUGATES

[0181] It is contemplated that the synthetic binding proteins may be engineered to modify certain desired properties (e.g., binding affinity, binding avidity, or pharmacokinetic or pharmacodynamic properties). This can be achieved by conjugating (e.g., chemical conjugation or via a fusion protein) a synthetic binding protein to an effector molecule. For example, the synthetic binding protein can be conjugated to a second binding molecule, e.g., a second synthetic binding molecule, which can be the same or different from the first synthetic binding protein, or an antibody or antibody fragment) or a molecule that directly (e.g., bovine serum albumin (BSA), murine serum albumin (MSA), or human serum albumin (HSA)) or indirectly (e.g., an engineered binding site for BSA, MSA, or HSA) enhances the PK or PD properties of the binding molecule. By way of non-limiting example, half-life can be extended by a variety of approaches known to those of skill in the art including use of polyethylene glycol (PEG), fusion proteins (e.g., Fc fusions, albumin fusions), engineered Fc binding such as engineered binding to neonatal Fc receptor (FcRn), antibody conjugation (e.g., to an antibody or fragment thereof), e.g., other protein engineering approaches that change the stability and / or clearance of a protein from an organism. It is contemplated that certain half-life extension approaches such as PEGylation may reduce clearance and prolong circulation. Fusions, such as to Fc or albumin, and / or addition of binding domains such as engineered HSA domains may prolong half-life due to circulatory half-life properties of the IPTS / 128584488.1 Page 91 of 180molecules (e.g., longer circulatory half-life via binding to albumin in the serum). For instance, a serum albumin binding domain may facilitate longer half-life via binding to albumin in circulating blood. One mechanism that extension could occur may include via recycling of a synthetic TNFR1 binding protein by binding to neonatal Fc receptor (FcRn), which could thereby extend the serum half-life of the synthetic TNFR1 binding protein.

[0182] It is contemplated that a variety of effector molecules can be used to modify the properties of the synthetic TNFR1 binding protein disclosed herein. Furthermore, it is contemplated that the effector may be a cytotoxic molecule or detectable label (e.g., radiolabel or fluorescent tag) which can be used in a detection assay, e.g., a diagnostic assay.

[0183] It is contemplated that the effector molecule can be chemically conjugated to the synthetic binding protein or can be incorporated into the synthetic binding protein as a fusion protein. The chemical conjugation can be accomplished by including a conjugation site into the synthetic binding protein, e.g., via inclusion of a derivatizable amino acid (e.g., a lysine or cysteine amino acid). The conjugation site can then be used to link the effector molecule to the binding protein, either directly or indirectly (e.g., via a linker, such as homobifunctional or a heterobifunctional cross-linking agent). By way of non-limiting example, linkers may be or include hydrazone, PEG, bifunctional 4-(4-acetylphenoxy) butanoic acid moiety, maleimidocaproyl, maleimidomethyl cyclohexane-1-carboxylate, maleimidocaproyl group with a tetrapeptide portion consisting of the amino acid sequence, glycine-glycine-phenylalanine-glycine, and / or maleimidocaproyl group with PEG. Exemplary homo- and heterobifunctional cross-linking agents can also include, for example, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), sulfo-NHS (N- hydroxysulfo succinimide), NHS (N-hydroxysuccinimide), dimethyl pimelimidate dihydrochloride, suberic acid bis(N-hydroxysuccinimide ester), DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), DSP (dithiobis(succinimidyl propionate)), sulfo- SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SPDP ((succinimidyl 3-(2-pyridyldithio)propionate), N-succinimdyl oxycarbonylethyl methanethio sulfonate, 6-Maleimidohexanoic acid N-hydroxysuccinimide ester, 6-Maleimidocaproic acid sulfo-N-succinimidyl ester, maleimidoacetic acid N-hydroxysuccinimide ester, N- succinimidyl iodoacetate, and 4-(4-maleimidophynyl) butyric acid N-hydroxysuccinimide ester.

[0184] Linkers may be used to link two or more synthetic TNFR1 binding proteins to one another, and may also be used to link one or more synthetic TNFR1 binding proteins to one IPTS / 128584488.1 Page 92 of 180or more effectors. A linker may be a peptide linker or a chemical linker. Linkers may be covalently bound (e.g., to an amino acid) to a synthetic TNFR1 binding protein and covalently bound to a second agent (e.g., an effector, e.g., a second binding protein). The composition and / or length of the linker may be designed with a particular functionality in mind, but preferably is non-immunogenic. A linker may contain one or more glycine amino acids and / or one or more serine amino acids. Exemplary linkers can comprise one or multiples of (Gly2Ser)n (SEQ ID NO: 1057), (Gly3Ser)n (SEQ ID NO: 1058), or (Gly4Ser)n(SEQ ID NO: 1059), where n can be 1, 2, 3 etc.

[0185] It is contemplated that the synthetic TNFR1 binding proteins disclosed herein can monovalent or multivalent. Multivalent proteins may include, but are not limited to bivalent and trivalent formats. A multivalent molecule may include two, three, four, or more monovalent synthetic TNFR1 binding proteins, wherein at least one linker connects a C- terminal amino acid of a first monovalent binding protein to an N-terminal amino acid of a second monovalent synthetic binding protein, such that the first and the second monovalent synthetic TNFR1 binding proteins are linked together. For example, in some embodiments, a multivalent (e.g., bivalent) miniprotein may comprise two miniproteins, each independently between about 35 and 85 amino acids and associated (e.g., linked, conjugated) with one another. In some embodiments, multivalent molecules may be fused and / or combined to another molecule, such as an effector molecule, e.g., a half-life extender (e.g., a site or a protein that binds to serum albumin to extend serum half life, etc.).

[0186] In certain embodiments, the disclosure provides a multivalent (e.g., bivalent) protein comprising a plurality of synthetic TNFR1 binding proteins disclosed herein. The multivalent protein can comprise a first synthetic TNFR1 binding protein and a second synthetic TNFR1 binding protein linked together through at least one linker. A linker (e.g., a glycine and serine containing linker (e.g., GGS), can connect a C-terminal amino acid of the first synthetic TNFR1 binding protein to an N-terminal amino acid of the second synthetic TNFR1 binding protein. Depending upon the circumstances, the multivalent binding protein can have a binding affinity stronger than the binding affinity of each synthetic TNFR1 binding protein alone. In certain embodiments, the multivalent protein comprises a synthetic TNFR1 binding protein having an amino acid sequence, wherein the amino acid sequence comprises an amino acid sequence set forth in Table 12. IPTS / 128584488.1 Page 93 of 180VII. PHARMACEUTICAL COMPOSITIONS

[0187] Once produced, a synthetic TNFR1 binding protein disclosed herein can be formulated into a pharmaceutical composition.

[0188] For therapeutic use, a synthetic TNFR1 binding protein disclosed herein is combined with a pharmaceutically acceptable carrier. Various carriers (e.g., diluents, excipients, etc.) used in formulating and preparing pharmaceutical compositions are known and / or readily accessible to those of skill in the art. Depending upon the circumstances, a carrier can include a liquid (e.g., a sterile liquid) or a solid. A carrier may be selected from or comprise water, aqueous solvents, non-aqueous solvents, dispersion media, surfactants, antioxidants, buffers, adjuvants, tonicity agents, stabilizers, bulking agents, lyoprotectants, metal ions, chelating agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. Typically a carrier is approved by United States Food and Drug Administration and meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or other International Pharmacopoeia. Suitable formulations for use in the present disclosure are found in see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020). For a brief review of methods for drug delivery, see, e.g., Langer (1990) SCIENCE 249:1527-1533. The resulting pharmaceutical compositions are suitable for administration to a subject (e.g., an animal, e.g., a mammal, e.g., a human).

[0189] A pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as IPTS / 128584488.1 Page 94 of 180polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, polyethylene glycol (PEG), sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020)).

[0190] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L- glutamate, poly (2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(−)-3- hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[0191] Depending upon the circumstances, a pharmaceutical composition may contain nanoparticles, or lipid droplets, e.g., polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED.1: 10-29).

[0192] Pharmaceutical compositions containing a synthetic TNFR binding protein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intraperitoneal, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. In certain embodiments, the synthetic peptide is administered by subcutaneous administration. IPTS / 128584488.1 Page 95 of 180

[0193] Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0194] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, polyethoxylated castor oil or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0195] Pharmaceutical formulations preferably are sterile. Formulations can be sterilized, for example, by methods appropriate to retain activity and stability of the synthetic TNFR1 binding protein included therein. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[0196] Depending upon the drug substance and formulation, the resulting dosage forms can be stable for extended periods of time, such as 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, or more, when the dosage form is a liquid or solid. The formulations can be stable at room temperature or higher. It is contemplated that the dosage form is stable at ambient conditions in PBS. Alternatively the dosage form is frozen (e.g., a liquid or a lyophilizate) and stable under appropriate temperatures such as, e.g., -20°C, -80°C).

[0197] Depending upon the circumstances, the dosage forms can be formulated as a unit dose, which can include, for example, about 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1 g, 1.5 g, 2.5 g, 5 g, or 10 g of the drug substance.

[0198] The compositions described herein may be administered locally or systemically. It is contemplated that the compositions described herein are generally administered by parenteral administration. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. In certain embodiments, the IPTS / 128584488.1 Page 96 of 180pharmaceutical composition is administered subcutaneously or may be administered intravenously, e.g., via intravenous infusion. In certain embodiments, it is contemplated that the synthetic constructs disclosed herein can be administered by systemic administration.

[0199] Generally, a therapeutically effective amount of active component, for example, a synthetic TNFR1 binding protein disclosed herein, is in the range of 0.1 mg / kg to 1000 mg / kg, e.g., 1 mg / kg to 100 mg / kg, e.g., 10 mg / kg to 500 mg / kg, e.g., 500 mg / kg to 1000 mg / kg. In certain embodiments, the effective amount is in the range of 15 to 50 mg / kg. In certain embodiments, the effective amount is 15 mg / kg. In certain embodiments, the effective amount is 30 mg / kg. In certain embodiments, the effective amount is 50 mg / kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the active component, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood- level or tissue-level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the synthetic peptide, and the disease, disorder, or condition being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. VIII. METHODS OF USE AND TREATMENT

[0200] The TNFR1 binding proteins described herein can be used in a variety of different approaches. For example, the binding proteins can be used in a method of targeting TNFR1. The method comprises contacting a cell that expresses TNFR1 on its cell surface with a composition comprising the synthetic TNFR1 binding protein disclosed herein. In addition the TNFR1 binding proteins described herein can be used to modulate TNFR1 activity. The method comprises contacting a cell that expresses TNFR1 on its cell surface with a composition comprising the synthetic TNFR1 binding protein disclosed herein. In each method, the TNFR1 binding protein or the multivalent protein comprising a TNFR1 binding protein further comprises an effector molecule. In each of the foregoing methods, the synthetic TNFR1 binding protein or the multivalent protein inhibits or reduces TNFR1 activity in the presence of a TNFR1 ligand (e.g., TNF ^) relative to TNFR1 activity in the IPTS / 128584488.1 Page 97 of 180presence of the TNFR1 ligand (e.g., TNF ^) but in the absence of the synthetic TNFR1 binding protein or the multivalent protein.

[0201] The TNFR1 binding proteins may be used in treatment of a disease, disorder, or condition mediated by TNFR1. TNFR1-signaling mediates various intracellular signaling activities downstream of TNFR1 binding a ligand (e.g., TNF ^), including those related to cell death (e.g., apoptosis). See, e.g., Toussirot and Aubin (2016) supra and Li, et al. (2017) supra.

[0202] In certain embodiments, the disclosure provides a method of decreasing TNFR1- mediated activity in a subject in need thereof. The method comprises administering to the subject an effective amount of the pharmaceutical composition comprising a synthetic TNFR1 binding protein or multivalent binding protein disclosed herein. As such, the compositions described herein can be used in treating inflammation in a subject in need thereof.

[0203] Exemplary diseases, disorders, or conditions that may be treated with the TNFR1 binding proteins disclosed herein include those such as inflammatory diseases and / or autoimmune diseases, including those thought be mediated or impacted by the TNFR1 axis. For example, the synthetic TNFR1 binding proteins disclosed herein may be used to treat inflammatory and / or autoimmune diseases. In some embodiments, a disease that can be treated using a synthetic TNFR1 binding protein is selected from Addison’s disease (e.g., autoimmune Addison’s disease), acute liver failure, acute pancreatitis, acute respiratory distress syndrome (ARDS), adult-onset Still’s disease, Alzheimer’s disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune polyendocrine syndrome, autoimmune thrombocytopenic purpura (ATP), axial spondyloarthropathy, Bechet’s disease, cachexia (e.g., inflammation-associated carcinogenesis and cancer cachexia), celiac disease, cerebral edema, cardiomyopathy, chronic atherosclerosis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic liver failure, chronic obstructive pulmonary disease (COPD), chronic pancreatitis, chronic rhinosinusitis, Churg-Strauss syndrome, Cogan syndrome, colitis-associated cancer, congestive heart failure, Crohn’s disease, Cystic fibrosis, cytokine release syndrome, Degos’ disease, dermatitis herpetiformis, dermatomyositis, dermatomyositis-juvenile, diabetic macrovasculopathy, diabetic retinopathy, discoid lupus, IPTS / 128584488.1 Page 98 of 180endometriosis, eosinophilic esophagitis, eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), essential mixed cryoglobulinemia, fibromyalgia, fibromyalgia- fibromyositis, fibrosis, glomerulonephritides, gout and gouty arthritis, graft-versus-host disease (GVHD), granulomatosis with polyangiitis (Wegener’s granulomatosis), Graves’ disease, Guillain-Barré syndrome, Hashimoto’s thyroiditis, hepatitis B, hepatitis C, hepatitis C-associated autoimmune diseases, hereditary angioedema, hidradenitis suppurativa, HIV (human immunodeficiency virus)-associated autoimmune diseases, Hidradenitis suppurativa, IgA nephropathy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), inclusion body myositis, inflammatory bowel disease, inflammatory bowel diseases, inflammatory myopathies, inflammatory syndrome following IL-2 administration, insulin- dependent diabetes mellitus, interstitial cystitis, interstitial lung disease, ischemic heart disease, Jarisch-Herxheimer reaction, juvenile chronic arthritis (Still’s disease), juvenile idiopathic arthritis (JIA), juvenile rheumatoid arthritis (JRA), Kawasaki disease, lichen planus, lichen sclerosus, loosening of prostheses, lupus erythematosus (systemic), Macular degeneration, mastocytosis, Ménière’s disease, microscopic polyangiitis, migraine, mixed connective tissue disease, mixed cryoglobulinemia, multiple myeloma, multiple sclerosis, multisystem inflammatory syndrome (MISC), myasthenia gravis, myelodysplastic syndromes, myocardial infarction, neuroinflammatory disorders (such as multiple sclerosis and neurosarcoidosis), neuropathic pain, non-alcoholic fatty liver disease (NAFLD), non- alcoholic steatohepatitis (NASH), non-radiographic axial spondyloarthritis, ocular inflammatory diseases (such as uveitis), oral lichen planus, orchitis, osteoarthritis, osteoporosis, Paget’s disease of bone, panciaticular vulgaris, paraneoplastic syndromes, pemphigus, periodontal disease, peripheral neuropathy, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis, post-streptococcal glomerulonephritis or IgA nephropathy, primary agammaglobulinemia, primary biliary cholangitis, primary sclerosing cholangitis, progressive renal failure, pseudogout, psoriasis (including plaque psoriasis), psoriatic arthritis, pyoderma gangrenosum, reactive arthritis, Reiter’s syndrome, relapsing polychondritis, respiratory syncytial virus (RSV) infection, rheumatic fever, rheumatic heart disease, rheumatoid arthritis, rosacea, sarcoidosis, SARS- COV-2-related inflammation, sciatica, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), sclerosing cholangitis, sepsis, Sjögren’s syndrome, spondyloarthropathies, stroke, steroid-dependent giant cell arteritis, Takayasu arteritis, temporal arteritis / giant cell arteritis, thoracoabdominal aortic aneurysm repair (TAAA), thrombotic thrombocytopenic purpura (TTP), TNF receptor-associated periodic syndrome IPTS / 128584488.1 Page 99 of 180(TRAPS), type 1 diabetes, type 2 diabetes, ulcerative colitis, undifferentiated connective tissue disease, uveitis, vasculitis (e.g., systemic vasculitis, vasculitis of one or more organ systems), vitiligo, Waldenström macroglobulinemia, Whipple’s disease, Wilson’s disease, Yellow Fever vaccination, and Zollinger-Ellison syndrome.

[0204] In certain circumstances, the compositions described herein can be used to treat a subject diagnosed as having rheumatoid arthritis, juvenile idiopathic arthritis, plaque psoriasis including pediatric plaque psoriasis, psoriatic arthritis, axial spondylitis including ankylosing spondylitis and non-radiographic axial spondyloarthritis, Crohn’s disease, ulcerative colitis including pediatric ulcerative colitis, uveitis, or Hidradenitis Suppurativa.

[0205] It is contemplated that therapy can be accomplished using a synthetic TNFR1 binding protein alone, as a monotherapy, or as part of a combination therapy. The combination therapy may include one or more additional agents or therapeutic approaches known to those of skill in the art for treating inflammatory and / or autoimmune diseases, and may have been previously used, be already ongoing, or added to a treatment for a subject in need thereof.

[0206] A subject may be evaluated, e.g., by a healthcare provider, before, during, and / or after treatment with a composition provided herein. Depending on the outcome of the evaluation, a treatment may be continued or ceased, treatment frequency or dosage may change, or the patient may be treated with a different synthetic TNFR1 binding protein. Subjects may be administered a composition comprising the synthetic TNFR1 binding protein for a discrete period of time according to dosage paradigms described herein, including, optionally, until the disease, disorder, or condition is treated.

[0207] Subjects that can be treated include those suspected as having, having, or at risk of having an autoimmune disease, disorder, or condition. The methods described herein may include a step of selecting a treatment for a subject in need thereof. The method includes (a) identifying (e.g., diagnosing) the subject with an autoimmune condition, and (b) selecting a synthetic TNFR1 binding protein as described herein, to treat the subject. Synthetic TNFR1 binding proteins administered in an effective amount to a subject in need thereof may result in one or more of (a) reduced auto-antibody levels, (b) reduced inflammation, (c) improved organ function (d) reduced pain, (e) decreased rate or number of relapses or flare-ups of the disease, (f) increased quality of life and any combinations of any of the above. IPTS / 128584488.1 Page 100 of 180

[0208] Certain autoimmune diseases can be treated by modulating, e.g., reducing, TNFR1- mediated signaling. For example, in certain diseases, the level of a TNFR1 ligand, such as TNF ^, is increased resulting in increased signaling and cell-death through TNFR1 signaling thereby causing symptoms such as inflammation and / or organ dysfunction seen in diseases such as, e.g., rheumatoid arthritis, psoriasis, ankylosing spondylitis, and Crohn’s disease. See, e.g., Toussirot and Aubin (2016) supra; Li, et al. (2017) supra; Kalliolias and Ivashkiv (2016) NAT. REV. RHEUMATOL. 12(1):49.

[0209] TNFR1-mediated signaling may be evaluated by detecting levels of one or more downstream molecules and / or cytokines. In certain embodiments, such molecules and / or cytokines are expressed at higher levels in a subject having or suspected as having an inflammatory and / or autoimmune disease.

[0210] The present disclosure provides methods of treating a subject in need thereof by administering an effective amount of the synthetic TNFR1 binding protein to the subject. The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. The phrase administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0211] In certain embodiments, a method or composition described herein, is administered in combination with one or more additional therapies, e.g., surgery, radiation therapy, or IPTS / 128584488.1 Page 101 of 180administration of another therapeutic preparation. In certain embodiments, the additional therapy may include an anti-inflammatory agent. Exemplary anti-inflammatory agents include, for example, small molecules and biologic immunomodulators. Exemplary small molecules include, steroids, methotrexate, NSAIDs, hydroxychloroquine, sulfasalazine, leflunomide tofacitinib, and baricitinib. Exemplary biological immunomodulators include, for example, a monoclonal antibody or an antibody fragment or fusion, including, for example, etanercept, infliximab, adalimumab, certolizumab (e.g., certolizumab-pegol), and golimumab. In certain embodiments, the additional therapy may include a combination of therapeutics of different classes. IX. KITS

[0212] Synthetic TNFR1 binding proteins of the present disclosure may be included as part of a kit. A kit may comprise a container comprising or consisting essentially of a unit of a pharmaceutical composition comprising a synthetic TNFR1 binding protein, instructions for use, and optionally, one or more agents (e.g., a buffer or diluent, if appropriate, to dissolve the binding protein or dilute a solution containing the binding protein), and a dispenser. A kit may include a label indicating the intended use of the contents of the kit. The contents of the kit may be used for treating, monitoring and / or diagnosing a subject in need thereof.

[0213] The present disclosure is further illustrated by the following examples which should not be construed as further limiting. EXAMPLES EXAMPLE 1: INITIAL SCREENING

[0214] This Example describes an initial in vitro screen of a synthetic protein library using yeast display to identify synthetic proteins capable of binding human TNFR1. The library was designed in silico and was screened for members having certain characteristics, such as binding to the target (TNFR1) at particular concentrations of target (e.g., 10 ^M). Once identified, certain synthetic binding proteins were synthesized for use in downstream screening and discovery processes.

[0215] A synthetic binding protein library having greater than 1 billion members was screened for TNFR1 binding by yeast display. Each binding protein contained an N- terminal extension of SEQ ID NO: 1026 and a C-terminal extension of SEQ ID NO: 1029 IPTS / 128584488.1 Page 102 of 180to facilitate expression and detection in yeast display. Synthetic proteins that bound to TNFR1 were isolated from non-binding proteins through iterative rounds of magnetic and fluorescent selection, using standard labeling and selection techniques (see, e.g., Chao et al., (2006) NAT. PROTOC. 1(2):755-68). Briefly, magnetic selection was performed using streptavidin-coated beads saturated with biotinylated TNFR1. The beads were used to isolate yeast displaying miniproteins that bound to TNFR1. Isolated cells were grown so that additional rounds of selection could be completed. For fluorescence-mediated selection, yeast cells were labeled with biotinylated TNFR1 at 1 ^M for a first selection round. Cells were then labeled with streptavidin-APC and cells displaying miniproteins that bound to TNFR1 were sorted on APC fluorescence (Sony SH800 cell sorter)Thank you. All cells that showed a greater fluorescent signal than cells not labeled with TNFR1 were isolated. These cells were then grown so that additional selection rounds under more stringent conditions including lower concentrations of soluble TNFR1 could be completed.

[0216] After multiple selection rounds the amino acid sequences of synthetic proteins that bound to TNFR1 expressed on yeast were determined using next-generation sequencing methods. Briefly, plasmid DNA encoding the miniprotein sequences was isolated from the selected yeast cells using standard DNA isolation techniques for yeast cells. PCR was then used to amplify specific DNA fragments that encoded miniprotein sequences and append necessary DNA elements to enable sequencing on an Illumina Mi-Seq instrument. Thousands of unique miniprotein sequences were identified from conditions where they showed binding at equal to or less than a concentration of 1 ^M TNFR1. Representative exemplary sequences were selected for further validation. Miniproteins encoded by each of these sequences were expressed and purified from E. coli. Binding of each miniprotein to TNFR1 was measured by surface plasmon resonance (SPR).

[0217] As part of the miniprotein preparation, the TNFR1 binding miniproteins were exposed to 4M urea at temperatures of at least 50℃. These proteins retained their properly folded three-dimensional structure as confirmed by circular dichroism spectroscopy. Separately, each of these miniproteins also retained their properly folded three-dimensional structure after heating to 95℃ as confirmed by circular dichroism spectroscopy (but not including urea exposure).

[0218] A miniprotein showing binding to TNFR1 was chosen as the basis to create a new library with greater than 1 billion variants of that miniprotein. This library was screened for binding to human TNFR1 using standard yeast surface display techniques. Miniprotein IPTS / 128584488.1 Page 103 of 180variants that bound to TNFR1 were isolated from non-binding miniproteins through iterative rounds of magnetic and fluorescent selection, using labeling and selection techniques as described earlier in this Example. Thousands of unique miniprotein amino acid sequences were identified, and Reference Miniprotein 8 (SEQ ID NO: 1) was selected for further optimization. EXAMPLE 2: INITIAL OPTIMIZATION OF TNFR1 MINIPROTEINS

[0219] This Example describes the initial optimization of TNFR1 miniproteins from a selected miniprotein from Example 1. The amino acid sequence of SEQ ID NO: 1 was redesigned to increase thermal stability and resulted in 10 new proteins, referred to as Reference Miniproteins 9-18 (SEQ ID NOs: 7-9 and 11-17) as shown in TABLE 3A. Each of Reference Miniproteins 9-18 had the same secondary structural arrangement of loop amino acids and alpha helix amino acids the parent. Miniproteins encoded by each of these sequences were expressed and purified from E. coli. Binding characteristics of each miniprotein to human TNFR1 were measured as shown in TABLE 3B, and binding activities (Ka, Kd, and KD values) of each miniprotein were determined using SPR.

[0220] The resulting ten Series 3 proteins were similar to one another (see TABLE 3C). The amino acid sequences of Reference Miniproteins 9-18 share about 70-98% identity with one another and about 65-80% identity with that of Reference Miniprotein 8. TABLE 3A. Exemplary Series 3 Miniprotein Reference Sequences Reference X R R R R R R R R RIPTS / 128584488.1 Page 104 of 180Reference Miniprotein SEQ ID NO Amino Acid Sequence R sReference KD (M) Ka (1 / Ms) Kd (1 / s) Interaction Miniprotein T1 / 2* (mins) *interactionol of a plurality of TNFR1 binding proteins remain bound to TNFR1. TABLE 3C. Percentage identity matrix Reference ii i0 1 2 3 4 5 6 7 8103993353IPTS / 128584488.1 Page 105 of 180Reference Miniprotein 8 9011121314151617181950EXAM

[0221] This Example describes in vitro chemical stability of variant Reference Miniproteins 9-18 (SEQ ID NOS: 7-9 and 11-17) using biophysical characterization after exposure to an exemplary chemical denaturants.

[0222] Each of these TNFR1 miniproteins validated in Example 2 were exposed to 4M urea. Each retained their properly folded three-dimensional structure after heating to 95℃ and then returning to 25℃ as measured by circular dichroism spectroscopy. Reference Miniprotein 18 (SEQ ID NO: 9) had increased thermal stability (FIGs.2A and 3A) retaining its conformation across a variety of conditions and maintaining binding across a range of temperatures as compared to the parent (FIGs.2B and FIG.3B) or Reference Miniprotein 11 (SEQ ID NO: 11) (FIGs.2C and FIG.3C). Reference Miniprotein 18 (SEQ ID NO: 9) was selected for further development based on its superior thermal stability and binding affinity as compared to those of the other miniproteins developed in this series (see TABLE 4). TABLE 4. Thermal and Chemical Stability characteristics Reference Thermal Stability (refold Chemical ThermalIPTS / 128584488.1 Page 106 of 180Reference Thermal Stability (refold Chemical Thermal Miniprotein properly after heating to stability in 4M stability vs. EXMINIPROTEINS BY AFFINITY MATURATION AND IN VITRO OPTIMIZATION

[0223] This Example describes optimization of human and mouse TNFR1 miniproteins.

[0224] A synthetic miniprotein library of greater than 1 billion variants of Reference Miniprotein 18 (SEQ ID NO: 9) was screened for binding to human TNFR1 and mouse TNFR1 using standard yeast surface display techniques. Each protein was engineered to contain an N-terminal extension of SEQ ID NO: 1026 and a C-terminal extension of SEQ ID NO: 1029 to facilitate expression and detection in yeast display. Miniprotein variants that bound to TNFR1 were isolated from non-binding miniproteins through iterative rounds of magnetic and fluorescent selection, using labeling and selection techniques known to those skilled in the art and as described in Example 1. Thousands of unique miniprotein sequences were identified. All Series 4 sequences were selected based on their ability to show binding to both human and mouse TNFR1 at 125 nM in the context of yeast display. Reference Miniproteins 19-26 having SEQ ID NOs: 30, 190, 197-199, 390, 878, and 956, as shown in TABLE 5A were generated via affinity maturation of Reference Miniprotein 18, and then selected for further validation. Miniproteins encoded by each of these sequences were expressed and purified from E. coli. Binding characteristics of exemplary miniproteins to both human TNFR1 (TABLE 5B) and mouse TNFR1 (TABLE 5C) were measured by SPR. TABLE 11 includes various exemplary Series 4 miniproteins that were made and showed binding to TNFR1 at a concentration of at 100 nM or less (i.e., displayed binding affinity to TNFR1 at a strength of 100 nM or greater binding).

[0225] The resulting Series 4 proteins (see TABLE 12 for complete list) were similar to one another. The amino acid sequences of Reference Miniproteins 19-26 share about 77-94% identity with one another and approximately about 87-91% identity with that of Reference Miniprotein 18 (SEQ ID NO: 9). IPTS / 128584488.1 Page 107 of 180

[0226] The amino acid sequences of Series 4 proteins corresponding to each consensus sequence were also highly similar within the entire Series and within each subgroup of Series 4 proteins corresponding to a Series 4.1 through 4.19 consensus sequence (see Table 5D). TABLE 5A. Exemplary Series 4 Miniprotein Reference Sequences With Human and Mouse Binding Affinities Reference Miniprotein SEQ ID NO: Amino Acid SequenceMiniproteins Reference Human KD (M) ka (1 / Ms) kd (1 / s) Interaction 1 / 2 )IPTS / 128584488.1 Page 108 of 180TABLE 5C. Binding Characteristics to Mouse TNFR1. Reference Mouse KD (M) ka (1 / Ms) kd (1 / s) Interaction T1 / 2 (mins) Miniprotein. g p y p SeriesIdentity range (aboutmin - max%)

[0227] Based on the resu s, eerence nproen ( Q NO: 30) was selected for further optimization. IPTS / 128584488.1 Page 109 of 180

[0228] The sequence was redesigned to increase thermal stability, which resulted in Reference Miniproteins 27-31 and 39-50 (SEQ ID NOs: 962-970, 972-976, and 978-980) as shown in TABLE 6A. Binding characteristics were determined including affinities for human and / or mouse TNFR1 using SPR (TABLE 6B).

[0229] The resulting Series 5 proteins (22 unique miniproteins) were similar to one another. The amino acid sequences of Reference Miniproteins 27-31 and 39-50 share about 72-98 % identity with one another and approximately about 74-98% identity with that of Reference Miniprotein 19. The miniprotein of SEQ ID NO: 878 was independently generated in each of Series 4 and Series 5. TABLE 6A. Exemplary Series 5 Optimized Miniprotein Reference Sequences Reference SEQ ID Amino Acid Sequence Protein NO:IPTS / 128584488.1 Page 110 of 180TABLE 6B. Binding characteristics of Exemplary Series 5 Miniproteins to TNFR1 Reference TNFR1 Species KD (M) ka (1 / Ms) kd (1 / s) Interaction T1 / 2 Protein Used in Binding (mins) EVITRO BINDING CHARACTERIZATION

[0230] This Example describes optimization of selected TNFR1 miniproteins. In this Example, Reference Miniprotein 43 (SEQ ID NO: 966) was selected for further optimization.

[0231] The sequence of Reference Miniprotein 43 was redesigned to increase thermal stability and retain high affinity for both mouse and human TNFR1, which resulted in Reference Miniprotein 51 (SEQ ID NO: 997). A synthetic miniprotein library of greater than 1 billion variants of Reference Miniprotein 51 (SEQ ID NO: 997) was screened for binding to human TNFR1 using standard yeast surface display techniques. Each binding protein was IPTS / 128584488.1 Page 111 of 180engineered to contain an N-terminal extension of SEQ ID NO: 1026 and a C-terminal extension of SEQ ID NO: 1029 to facilitate expression and detection in yeast display. Miniprotein variants that bound to TNFR1 were isolated from non-binding miniproteins through iterative rounds of magnetic and fluorescent selection, using labeling and selection techniques known to those skilled in the art and as described in Example 1. This resulted in Reference Miniproteins 52-74 (SEQ ID NOs: 998-1020) as shown in TABLE 7A. Binding characteristics were determined including affinities for human (TABLE 7B) and / or mouse TNFR1 using SPR (TABLE 7C).

[0232] The resulting Series 6 proteins (24 unique miniproteins) were similar to one another. The amino acid sequences of Reference Miniproteins 51-74 share about 91-98 % identity with one another and approximately about 87-98% identity with that of Reference Miniprotein 43. TABLE 7A. Exemplary Series 6 Optimized Miniprotein Reference Sequences Ref. Protein SEQ ID NO: Amino Acid SequenceIPTS / 128584488.1 Page 112 of 180Ref. Protein SEQ ID NO: Amino Acid Sequence 67 1013 SARDYLRRLRDEGYISYNLEGQLNDLLDIGEDEQAVIDYALDFIESEReference Protein ka (1 / Ms) kd (1 / s) Human KD (M) 1 1 9E 06 285E 03 159E 09IPTS / 128584488.1 Page 113 of 180Reference Protein ka (1 / Ms) kd (1 / s) Human KD (M) 72 2.48E+06 1.72E-03 6.95E-10Reference Protein ka (1 / Ms) kd (1 / s) Human KD (M) 51 1.53E+06 7.24E-04 4.72E-10 EXAMPLE

[0233] This Example describes identification and characterization of a conserved paratope in TNFR1 miniproteins. IPTS / 128584488.1 Page 114 of 180

[0234] Site saturation mutagenesis (SSM) data were generated for Reference Protein 51 (SEQ ID NO: 997). The paratope was identified manually with SSM data derived from the Series 6 sequence, SEQ ID 997. That is, reference Miniprotein 51 (SEQ ID NO: 997) was used as a comparator and using yeast surface display of 19 natural amino acids (alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) other than cysteine, binding to TNFR1 was measured. Paratope residues were identified as occurring at positions 22, 26, 31, and 39 within the 47-mer of Reference Protein 51. Each position was substituted with each of the other 18 other naturally occurring amino acids (excluding cysteine), and binding was assessed using yeast display assays.

[0235] Briefly, each miniprotein being assessed was displayed on the surface of yeast at approximately 50,000 copies per yeast cell, and soluble TNFR1 was titrated against the yeast cells displaying the miniprotein at varying concentrations of TNFR1 from 10 pM to 1 ^M. Each binding protein was engineered to contain an N-terminal extension of SEQ ID NO: 1026 and a C-terminal extension of SEQ ID NO: 1029 to facilitate expression and detection in yeast display, resulting in miniproteins as set forth in SEQ ID NOs: 1033- 1056. These data were used to determine binding affinities of the miniproteins for TNFR1.

[0236] Paratope residues were identified at positions corresponding to amino acid residues relative to SEQ ID NO: 997, which is a 47-mer, namely at positions 22, 26, 31, and 39. Certain paratope residues were found to be immutable in that they were completely intolerant to substitution without loss of function (see, e.g., TABLE 8). TABLE 8. Paratope Residues and Tolerances Position Original Amino Acids1Tolerable Substitutions1 Amino acid identities at positions in SEQ ID NO: 993. Paratope was determined using SSM data with Reference Miniprotein 51 (SEQ ID NO: 997, which is a Series 6 protein of consensus sequence SEQ ID NO: 993) as a starting sequence; position refers to linear, ordinal position with amino acid position 1 being the N-terminal amino acid in SEQ ID NO: 993 and amino acid 47 being the C-terminal amino acid in SEQ ID NO: 993. IPTS / 128584488.1 Page 115 of 180Position Original Amino Acids1Tolerable Substitutions

[0237] The 3 or SEQ ID NO:1021 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 47 as the C-terminal amino acid) was 100% conserved over greater than 900 sequences across all series of miniproteins as provided herein in SEQ ID NOs: 1, 6-17, 26-48, 53-73, 78-147, 152-192, 197-271, 276-344, 349-366, 371-403, 408-444, 449-522, 527-562, 567- 602, 607-675, 680-737, 742-785, 790-810, 815-873, 878-895, 900-957, 962-982, 997-1020. Position 22 was 100% conserved across the entirety of Series 6 Miniproteins (SEQ ID NOs: 997-1020) and using SSM analysis of SEQ ID NO: 993 or SEQ ID NO: 1021 was found to be immutable in that it could not be changed from its identity as a glutamine (Q) without materially decreasing TNFR1 binding potency. In this example, a decrease in binding potency was considered a material decrease in binding potency when a TNFR1 binding protein had a binding affinity of weaker than 250 nM. The amino acid corresponding to position 26 of SEQ ID NO: 993 or SEQ ID NO: 1021 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 47 as the C-terminal amino acid) was 100% conserved in SEQ ID NOs: 997-1020 and using SSM analysis of SEQ ID NO: 997, could only tolerate substitution from leucine (L) to isoleucine (I), alanine (A), or valine (V) without materially decreasing TNFR1 binding potency to greater than 250 nM as measured by yeast on-cell affinity. The amino acid corresponding to position 31 of SEQ ID NO: 993 or SEQ ID NO: 1021 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 47 as the C-terminal amino acid) was 100% conserved in SEQ ID NOs: 997-1020 and using SSM analysis of SEQ ID NO: 997 could only tolerate a substitution from glutamic acid (E) to aspartic acid (D) without materially decreasing TNFR1 binding potency. The amino acid corresponding to position 39 of SEQ ID NO: 993 or SEQ ID NO: 1021 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 47 as the C- terminal amino acid) was 100% conserved in each of SEQ ID NOs: 997-1020 and using SSM analysis of SEQ ID NO: 997, could only tolerate substitution from tyrosine (Y) to phenylalanine (F) without materially decreasing TNFR1 binding potency.

[0238] A conformational paratope of a TNFR1 binding protein of the present disclosure can be represented as X22 - X26 - X31 - X39, where X22 is Q, X26 is L, X31 is E, X39 is Y. Without losing binding potency as measured by binding at 11 nM or stronger, X26 could also IPTS / 128584488.1 Page 116 of 180be V, I, or A, X31 could also be D, and X39 could be F. X22 could not be substituted without loss of potency beyond the threshold. EXAMPLE 7: DESIGN & SYNTHESIS OF BIVALENT TNFR1 MINIPROTEINS

[0239] This Example describes the design, production, and binding characterization of bivalent TNFR1 miniproteins.

[0240] Exemplary Reference Miniprotein amino acid sequences were converted into bivalent sequences by taking the entire amino acid sequence of each of Reference Miniproteins 23, 24, 25, 26, and those of SEQ ID NOs: 324, 417, and 783 (i.e., monovalent Reference Miniproteins) to generate bivalent Reference Miniproteins 32-28. Briefly, bivalent miniproteins were designed by taking each of the monovalent Reference Miniproteins, adding a three residue glycine, glycine, serine (GGS) linker to either the N-terminal side or C- terminal side of a given Reference Miniprotein, and then adding the entire amino acid sequence of each of the same Reference Miniprotein as the GGS was added to onto whichever side (N- or C-terminal side) of the GGS linker is exposed and available for association. The short linker was designed to prevent the bivalent miniproteins from crosslinking TNFR1 receptors (e.g., in a cell), which could risk conferring agonist function onto a given bivalent molecule. Amino acid sequences of Reference Miniproteins 32-38 are set forth in TABLE 9A. Reference Miniproteins 32-38, containing the amino acid sequences of SEQ ID NOs: 983-989, respectively, were expressed and purified from E. coli. TABLE 9A. Exemplary Multivalent Synthetic Miniproteins Reference SEQ ID Amino Acid Sequence Derived From :IPTS / 128584488.1 Page 117 of 180Reference SEQ ID Amino Acid Sequence Derived From Protein NO SEQ ID NO:and / or mouse TNFR1 using SPR (TABLE 9B) TABLE 9B. Binding characteristics to TNFR1 Reference TNFR1 Species Used Human ka (1 / Ms) kd (1 / s) Interaction Protein in Binding Studies TNFR1 KD T1 / 2 (mins) EXAFR2

[0242] This Example describes the characterization of binding specificity of TNFR1 miniproteins engineered, developed, and produced in accordance with Examples 1-5.

[0243] During yeast display screening, Reference Miniproteins that bound TNFR1 but not TNFR2 were isolated and characterized. Binding was measured via SPR. As shown in TABLE 10, Reference Miniproteins 23 and 26 (SEQ ID NOs: 878 and 390) did not show binding to TNFR2 at all, while binding to TNFR1 was at nM concentrations. Bivalent Reference Miniproteins 32 and 35 (SEQ ID NOs: 983 and 986) each bound TNFR2, but at much lower affinity than compared to TNFR1, and also bound to TNFR1 more tightly than monovalent Reference Miniproteins 23 and 26. IPTS / 128584488.1 Page 118 of 180TABLE 10. TNFR1 and TNFR2 Binding Reference SEQ ID NO: TNFR1 KD TNFR2 KD Protein EXAMPLE 9: I OTEINS

[0244] This Example demonstrates the ability of TNFR1 miniproteins to inhibit TNF signaling in vitro. Reference Miniproteins 23 and 32 were tested in assays that measure inhibition of TNF signaling in either mouse or human cells. Reference Miniproteins 51, 52, 57, 62, 69, 71, 73, and 74 were also tested in assays that measure inhibition of TNF signaling in human cells. L929 Assay (mouse cells)

[0245] The efficacy of TNFR1 miniproteins to influence TNF signaling in mouse cells was tested by measuring viability of cells after exposure to TNF ^ in the presence of a control miniprotein (a miniprotein that does not bind to TNFR1) or to each of two Reference Miniproteins known to bind to TNFR1. L929 cells are mouse origin cells and a standard cell type in which to test TNF signaling. The TNF activity assay was performed in accordance with methods known to those in the art.

[0246] Briefly, various concentrations of miniproteins, ranging from 0.001 nM to 1,000 nM in 10-fold increments, were combined with L929 cells and incubated for 15 minutes at 37℃. Then, fixed concentrations of mouse TNF ^ and Actinomycin D were added to the pretreated L929 cells. After 16 hours of culture at 37℃, cell viability was measured using luminescence. The greater the number of viable cells, the greater the luminescent signal in RU. As shown in FIG.5, cells treated with a control miniprotein showed low viability (flat line) at all concentrations tested. In contrast, cell viability in the presence of Reference Miniproteins 23 and 32 was increased, demonstrating that each of these miniproteins were effective at nanomolar or picomolar concentrations in protecting cells from death after exposure to TNF ^. The IC50 of Reference Miniprotein 23 was 3.237 nM and the IC50 of Reference Miniprotein 32 was 212.2 pM. IPTS / 128584488.1 Page 119 of 180HEK assay (human cells)

[0247] Efficacy of TNFR1 miniproteins to influence TNF-mediated signaling in human cells was tested by measuring TNF-pathway activity in human embryonic kidney cells (HEK) modified to express a secreted reporter that is a surrogate of TNF activation in the presence of a control miniprotein (a miniprotein that does not bind TNFR1) or a Reference Miniprotein known to bind TNFR1.

[0248] Briefly, various concentrations of miniproteins, ranging from 0.0001 nM to 10,000 nM in 10-fold increments, were combined with the modified HEK cells incubated for 15 minutes at 37℃. Then, fixed concentrations of human TNF ^ were added to the pretreated HEK cells. After 16 hours of culture at 37℃, supernatant was collected and measured to determine TNF ^-dependent reporter activity (in accordance with methods known to those in the art). As shown in FIG.4, cells treated with a control miniprotein (triangle icons on graph) at 10, 100, or 1000 nM showed no inhibition of TNF activity. In contrast, TNF activity in the presence of Reference Miniprotein 19 decreased as concentration of the miniprotein increased. A sharp decrease in TNF-mediated signaling occurred between concentrations of 1 nM and 10 nM of Reference Miniprotein 19. At concentrations greater than 100 nM, greater than 99% of TNF activity was neutralized. The IC50of Reference Miniprotein 19 was 6.33 nM. For Reference Miniprotein 32, at concentrations greater than 10 nM, greater than 99% of TNF activity was neutralized. The IC50of Reference Miniprotein 32 was 0.643 nM. Reference Miniproteins 51, 52, 57, 62, 69, 71, 73, and 74 (SEQ ID NOs: 997, 998, 1003, 1008, 1015, 1017, 1019, and 1020, respectively) were also each verified to inhibit TNFR1 signaling in the HEK-cell-based assay. IC50 values are shown in TABLE 11. TABLE 11: IC50Values of Exemplary Reference Miniproteins Reference Miniprotein SEQ ID NO IC50IPTS / 128584488.1 Page 120 of 180Reference Miniprotein SEQ ID NO IC50 74 1020 6.08E-09 EXAMPLE 10: IN S

[0249] This Example describes in vivo inhibition of TNF ^-mediated toxicity using TNFR1 miniproteins as compared to an exemplary TNF ^ antibody. TNFR1 miniproteins prevented death after administration of a normally lethal dose of TNF ^.

[0250] Briefly, six-week old male C57 / BL6 mice (n= 8 per condition) were treated with a test agent selected from a control miniprotein, a TNF ^ antibody, or one of two TNFR1 Reference Miniproteins. All mice were injected with 50 ^L of test agent by intravenous (i.v.) tail vein injection. Fifty microliters of 80 ^M TNF ^ (a normally lethal dose, i.e., greater than the LD100) were i.v. injected into each mouse. Mice in the miniprotein control group were injected 20 minutes prior to the TNF ^ ^injection. Mice in the TNF ^ antibody group were injected with test agent 60 minutes prior to TNF ^ injection. Mice in the Reference Miniprotein test groups were injected with miniprotein either 20 minutes prior, at the same time as, or 20 minutes after TNF ^. Survival was monitored for 48 hours.

[0251] As seen in FIG.6, all mice in the miniprotein control group died 16 hours after TNF ^ injection. Eighty percent (six of eight) mice in the TNF ^ antibody group died by 24 hours post TNF ^ injection; the remaining two were still alive by 48 hours. In contrast, all mice treated with either Reference Miniprotein 23 or 32 were alive, regardless of whether they were treated prior to, at the same time as, or after administration of the TNF ^ dose, demonstrating that TNFR1 miniproteins protect against toxic TNF ^-mediated signaling. That is, TNFR1 miniproteins provided complete protection against TNF ^-induced lethality at all administration time points tested. INCORPORATION BY REFERENCE

[0252] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety for all purposes. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. IPTS / 128584488.1 Page 121 of 180EQUIVALENTS

[0253] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. IPTS / 128584488.1 Page 122 of 180LVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVSIEILVLVLVLVLVL L L L L L L LY Y Y H D D G Y D D D D D D H H R Y YV V V V V V V VS S S S D S S S S S S S S S S SG G G G G G G G G G G GI I I I I I I I I I IG D D S S S S S S S S S S S S SR R G R Y R RI I I I I E L 1 I I I I I I I I I I I I IG G G G G G GRGRGRGRRRGRGRGAGLGDRLD4XRGRGRGRGRGRGRGRGRGRGRGRGRE E E E E E E E E E E E S F E E L D I A E E E E E EGD D D D D K S S L K D D K D D D R R L Y DE E E E E E EE CR R R R R R R R R R R R R RD D D D D D D D D D D DLRLRLRLRLRLRLRLRLR R 7 L Q D R R R R R R R R R R R R RRLRLRLRLRLRLRLRXLR7Q0IVLRLRLRLRLRLRLRLRLRLRL L LNE E E K E E R E E F E E E E E K Y X 2 A E E E ER R REL L L L L L L L L L L L L L L L 4 LE E E E E E E E EUYAYEYKYDYEYDY Y Y Y Y Y Y G Y Y X YXL43LYLYLYLYLYLYLYLYLYLYLYLYLY QD D Q R R RERERERERDYSKDRDRIRDRRA4XV7X3DRDRDRERERDRDRD E D D D DE A A A A A A A A A A A A A A ASAL1XRA1X3R R R R R RS S S S R S S S S S S S S D S XASASASASASASASASASASAFAVARE2.4r 2.24.2.Me4 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KUYDYDYDYDYLYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLQYLDQLDRLTYLTYLEYLYLYLYLYLYLYLYLYLYLYLY QR R R R R R R R R R R R R R RF D D D D D D D D D D DE A A ATASASAIAVAR R R R R R R R R R R R R R R R R RS S S KAHADASASAQASARASASASASASASASASASASAIASASASASASASASEM A N / SEIR E3.3.3.3 3 3 3 3 3 3 3 3 3 3 34.4.4.4.4.3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3S 4 4 4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4:O NDI Q E90010203040506 7 8 9 0 1 2 3 4 5 6 710101010118 9 0 1 2 3 4 5 6 7 8 9 0 1S 9 1 1 1 1 1 1111111111111111111212121212121212121213131IPTS / 128584488.1 Page 126 of 180LVLVLVLVLVLVLVLVLVLVLVLYLVLVLVLVLVLFLVLVLVL L L L L L L L L L L LD D S F Y Y Y Y Y Y Y H Y Y HV V V V V V V V V D V VS S S S S S S S S SY Y H H Y A G Y Y F R F G G R R Y NI I I I I I I I I ISISISISISISISISISISISISISISISISISISISISISISISR H F R R R R R R R R R R R F R R R R R K H I T Y RIG G G G G H G G G G G G G G G G G G G G GF R R R R E RT E E N F E E E E E E E G S EG G G G G G G G G G G GD D D D D D D D D DE E E E E E E E E E E E F E E E E EER R R R R R R R R RDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRD DCLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRL L L L L L L L L L L LRLRL NE E E E E E E E E K K ER R R R R R R R R R R R R EEL L L L L LE E E E E E E E E E E E E E E E Q G E E EUYDYDYDYDYDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLYLYLYLYLYLYLYLYLYLYLY QR R R R R R D A H R R R R R RD D D D D D D D D D D DE A A ASASASASASAR R R R R R R R R R R R R R R R R RS S S SASASASASASASASAVAYASAFAKASASASASASARASASASASASASASEM A N / SEIR E5.5.5.5 5 5 5 5 5 5 5 5 5 5 54.4.4.4.4.5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5S 4 4 4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4:O NDI Q E8999001020304 5 6 7 8 9 0 1 2 3 4 520202020206 7 8 9 0 1 2 3 4 5 6 7 8 9 0S 1 1 2 2 2 202121212121212121212122222222222222222222232IPTS / 128584488.1 Page 129 of 180LVLVLVLVLVLVLVLVLFLTLTLTLVLVLVLVLVLVLVLVLVL L L L L L L L L L L LY E G Y Y Y A Y F Y Y D H H HV V V V V V V V V V V VS S S S S S S S S SS R F F F F F F H Y Y F T H D Y Y SI I I I I I I I I ISISISISISISISISISISISISISISISISISISISISISISISQ Q F R Q F R R R R R R R R R R R R R R R R R R R RIG G G G G G G G G G G G G G R G G G K G GR R R R R R RE E E E E E E E E E E Y E E EG G G G G G G G Q G G GD D D D D D D F D DE E E E E E E E E E E E E V E E E EER R R R R R R R R RDRDRDRDRDRDRDRDRDRDRDRDRIRDRDRDRDRKRDRDRDRD DCLRLRLRLALRLRLRLRLRLRLRLRLRLRLRLRLRLRLRL L L L L L L L L L L LRLRL NE E E E E E E E E E E ER N Q R R R R R R R R R R REL L L L L LE E E E E R E E E E E E E E E E E R L I EUYDYDYDYDYDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLRYLRYLDYLDYLDYLDYLYLYLYLYLYLYLYLYLYLYLY QR R R R R R R R R R R R N Q RD D N D D D D D D D D DE A A ASASASASASAR R R R R R R R R T R R R R R R R RS S S SASASASASASASASASASASASASASASASASASASAGASASASASASASEM A N / SEIR E5.5.5.5 5 5 5 5 5 5 5 5 5 5 54.4.4.4.4.5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5S 4 4 4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4:O NDI Q E1323334353637 8 9 0 1 2 3 4 5 6 7 823232324249 0 1 2 3 4 5 6 7 8 9 0 1 2 3S 2 2 2 2 2 242424242424242425252525252525252525262626262IPTS / 128584488.1 Page 130 of 18074 X 6LVLVLVLVLVLVLVLVX011X4XDLVLVLVLVLVLTLVLVL L L L L L L L L L L L LD H H H H H F F 1 X 2 1 F F F FT V V V V V V V V V V V VS S S S S S S S X 0 X 4D Y H K S R H V D F D Y Y Y Y Y YI I I I I I I IS S S S D S S S S S S S S S S S S S S S SR Y R RL 1 L X I I I I I I I I I I I I I I I I I I I I IG G G GRGRGRGRG8XXLQ1AYRGRGRGRGRGRGRKRGRGRGRGRGRGRGRGRGFGRGR R RE E E E E E E E 7 8 2 D E E E E E E E F E R G E E EG G GD D D D D D D D X X X 7 D D D D D D D D D DE E E E E Y EE CR R R R R R R R L 7 0 3 R R R R RD D D D D D D D D D DLRLRLRLRLRLRLRLR5 X 2 X L L L L LRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRL NE E EX L X V R R R R R R R R R R R R R R R R R R R R REL L LELELELELEL4X5XL8A4ELRLELELGLELELELELELELELELELTLELE E E E EUYDYDYDYDYEYQYDYD3X4X1X3XYQYEYFYDYDYDYDY Y Y Y T Y Y Y YLYLYLYLYLY QR R R R R R R R A 3 7 3 R RD D D D D K D D D D D D D DE A A A A A A A A 1Y A R R R R R R R R R R R R R R S R RS K S S S S S K A XXA1X3XASASASARASAKASASASASASASASASASASASASASASAFE6.4Mr 6e.6 64.4.4ASrN / _ereresS S SSuEs _In 1_ _e n2in3nsaiaiaR E5.5.5.5.5.5.5.5.no momomo 6.6.6.6.6.6.6.6.6.6.6.6.6.6.6.6.6 6 6 6 6S 4 4 4 4 4 4 4 4 CD D D4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4.4.4.4.4.4:O NDIeE66 7 8 9 0 1 2neQ4 5n526262626o o 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6S 262727272N N 72727272728282828282828282828292929292929292IPTS / 128584488.1 Page 131 of 180LVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVL L L L L L L L L L L LY Y D D Y Y Y Y Y K D H A A AV V V V V V V V V V V VS S S S S S S S S SH G Y Y Y H H H H H S A A A H Y H HI I I I I I I I I ISISISISISISISISISISISISISISISISISISISISISISISR H R R R R R R R R R R R R R R R R R R R R R R R RIG G G G G G G G G G G G G G G G G G G G GR R R R R R RE E E E E E E E E E E E E E EG G G G G K G G G N G GD D D D D D D D D DE E L E E E E E E E E E E E E E E EER R R R R R R R R RVRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRDRD DCLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRL L L L L L L L L L L LRLRL NE L H R E E F K R R E ER R R R R R R R R R R R R REL L L L L LE E E E E E E R L A R G I L E G G V E E EUYDYDYDYDYDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLDYLYLYLYLYLYLYLYLYLYLYLY QR R R R R R R R R R R R R R RD D D D D D D D D D D DE A A ASASASASASAR R R R R R R R R R R R R R R R Q GS F S SASASASASASASASASASASASASASASASASASASASASASASASASASEM A N / SEIR E6.6.6.6 6 6 6 6 6 6 6 6 6 6 64.4.4.4.4.6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6S 4 4 4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4.4:O NDI Q E7989990010203 4 5 6 7 8 9 0 1 2 3 430303030305 6 7 8 9 0 1 2 3 4 5 6 7 8 9S 2 2 2 3 3 303031313131313131313131323232323232323232323IPTS / 128584488.1 Page 132 of 180LVLVLVLDLVLVLVLVLVLVLVLVLVLILVLVLVLVLVLVLVLVLVL L L L L L L L L LK A R T H H H Y Y H Y Y Y Y Y Y Y YV V V V V V V V V VS S S S S S S S S S S S S SY Y A A A Y Y Y Y G Y F Y Y YI I I I I I I I IS S S S S S S S S S S S S S S S S S SR R R R RI I I I I I I I I I I I I I I I I I I I I I I IN G G G GRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGR E R RE K N E E E R E E E E E E E L L L E F N E E E EG G G GD D D D D D D D D D D D D D D D D D D DE E E E E E E E EE CR R R R R R R R R R R R R RD D D D D D D F D D D D DL L L L L L L L L LR R R R R R R R R R V R R R R R R R RN R R R R R R R R R RLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLR EELELELELELELELELELRLRLELELELELELELELELELGLGLELE E E I E E E E E EUYDYDYDYDSDEDYDVDVDYDYDFDYDY Y Y Y Y Y Y Y Y YLYLYLYLYLYLYLYLYLYLY QR R R R R R RD D D D D D D D D D S K I D D L E D D TE A AK Q R R R R R R R R A R R R R R R R R R R R R R R RS S SASASASASASASASASASASAQASASASASASASASASASASASASASASASASASASASASEM A N / SEIR E010.10.10.10.10.10.10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1 1 1S 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4.4.4.4:O NDI Q E263646566 7 8 9 0 1 2 3 4 5464646466 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4S 4 4 4 474747474747474747474848484848484848484849494949494IPTS / 128584488.1 Page 137 of 180LVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVL L L L L L L L L LF N K Y Y Y G F H H H Y H Y Y Y S YV V V V V V V V V VS S S S S S S S S S S S S SY T H H H A F Y Y F G R S H NI I I I I I I I IS S S S S S S S S S S S S S S S S S SR R R R RI I I I I I I I I I I I I I I I I I I I I I I IG G G G GRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRRRRRRRGR R R RE E G E S E E E E E E E E E E E E E E E E E E EG G G GD D D D D D D D I D D D D D D D D D D DK E E E E E E E EE CR R R R R R R R R R R R R RD D D D D D D D D D D D DL L L L L L L L L LR R R R R R R R R R R R R R R R R R RN R R R R R R R R R HLRLRLRLVLHLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLR EELELELELELELELELELELELELELELELELELELELELELELELE E E E E E E E E EUEDEDYDYDYDYIYIYNYDYDYHYHYLY Y Y Y Y Y Y Y Y YLYLYLYLYLYLYLYLYLYLY QR R R R R R RD D D L D D K D A D D D D D D D D D D DE A AR R R R R R R R R R R R R R R R R R R R R R R R R RS S SASASASASASASASASASASASASASASASAKASASASASASASASASASASASASASASASEM A N / SEIR E111.11.11.11.11.11.11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1 1 1S 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4.4.4.4:O NDI Q E829203132 3 4 5 6 7 8 9 0 1535353532 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0S 5 5 5 535353535454545454545454545455555555555555555555565IPTS / 128584488.1 Page 139 of 180LVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVL L L L L L L L L LD D D D D D D D D D D D D D D D D DV V V V V V V V V VS S S E S S S S S S S S S SD D D D D K D D D D D D D D DI I I I I I I I IS S S S S S S S S S S S S S S S S S SR R R R RI I I I I I I I I I I I I I I I I I I I I I I IG G H G GRGRGRGRGRGRGRGRGRKRGRGRGRGRGKGRGRGRGRGRGRGRGRGRGR R R RE E E E E T T E E E G E E Y E E E E E E E E E VG G R RH D D D D D D D Q D D I D D D D D D D DE E Y E E E E E EE CR R R R R R R R R V R R R RD D D D D D D D D D D N FL L L L L L L L L LR R R R R R R R R R R R R R R R R R RN R R R R R R R R R RLRLRLRLRLRLRLRLLLQLRLRLKLRLRLALRLRLRLRLNLRLRLR EELELELELRLELRLELELELELELELELELELELELELELNLELALE E E E E E S E E EUYDVDEDYYYYGDYDYDYDYDYDYDVDY Y Y Y Y Y Y Y V SLYLYLYLYLYLYLYLYLYLY QR R R R R R RD D D D D D D D D D D D D D A L D G D DE A AS R R R R R R R R R R R R R R R R R Y R R R R R R RS S SASASASASASASASASARASASASASAKAIASASASASASASASASARASAQARASASASASEM A N / SEIR E313.13.13.13.13.13.13 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1 1 1S 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4.4.4.4:O NDI Q E728292031 2 3 4 5 6 7 8 9 0636363631 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9S 6 6 6 636363636364646464646464646464656565656565656565656IPTS / 128584488.1 Page 142 of 180LVLVLVLVLVLVLVLDLDLVLVLVLVLVLVLVLVLVLVLVLVLVLVL L L L L L L L L LD D D D Y D D D D D D D D D D D D DV V V V V V V V V TS S S S S S S S S S S S S SD D D D D D D D D D D D D D DI I I I I I I I IS S S S S S S S S S S S S S S S S S SR R R R RI I I I I I I I I I I I I I I I I I I I I I I IG G G G GRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRGR R R RT E E R S S E N Y T E G E E L G E E E E E E R EG G G GD D D D D D D D D D D D D D D D D D D DQ F E E E E E E EE CR R R R R R R R R R R R R RD D D N D D D D D D D D DL L L L L L L L L LR R R R R R R R R R R R R R R R R R RN R R F Q R R R R R RLRLRLRLRLRLRLRLTLTLFLFLRLRLRLRLRLKLKLYLYLYLRLR EELELELELELELYLELELELELELQLELELELELELALELELELELE E E E E Q E E E EUYDYFYDYDYEYLYYYDYDYAYYYFYYY Y Y Y Y Y Y Y Y YLALILVLGLELYLVLVLHLY QQ Q R R R R RY D D D D D D D T D D D D D D D D D D DE A AR R R R R R V R R R R R G R R R R R R R R R R R R RS S SAGASASASASASASASASASASASASASAEAEASASASASASASASASASASASASASASASEM A N / SEIR E414.14.14.14.14.14.14 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1 1 1S 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4.4.4.4:O NDI Q E394959697 8 9 0 1 2 3 4 5 6696969607 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5S 6 6 6 707070707070707070717171717171717171717272727272727IPTS / 128584488.1 Page 144 of 180LVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVL L L L L L L L L LD D D D D D D D D D D D D D D D D DV V V V V V V V V VS S S S S S S S S S S S S SD D D D D D D D D D D D D D DI I I I I I I I IS S S S S S S S S S S S S S S S S S SR R R R RI I I I I I I I I I I I I I I I I I I I I I I IG G G G GRGRHRGRGRGRGRGRGRGRGRGRGFGYGYGYGHGYGYGYGFGRGRGHGS R F RQ E I E E E E E E E E E E H I F E E L E E A F EG G G GR R D D H D D D D D D D D D D D D D D DE E E A E E Y E EE CR R R R R R R R R R Y R R RD D D D D D D D D D D D RL L L L L L L L L LR R R R R R R R R R R R R R R R R R RN R R R R T Y Y T R RLRLRLRLRLRLRLYLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLR EELLLELKLELELELELSLELELELSLELRLELGLELELELELELELE E E A Q E E K R EUYDYDYDYDYDYDYDIDSDYDYDADYDY Y Y Y Y Y Y Y Y YLYLYLYLYLYLLLTLYLYLY QR R R R R R RD D D D D D D D D D D D D D D D D D D DE A AR R R R R R R R R R R R R R R R R H R Y R R R R R RS S SAHAGASASASASASALASASASGSASASASASASASASASASASASASASASASASASASASEM A N / SEIR E717.17.17.17.17.17.17 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1 1 1S 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4.4.4.4:O NDI Q E526272829 0 1 2 3 4 5 6 7 8828383839 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7S 8 8 8 838383838383838484848484848484848485858585858585858IPTS / 128584488.1 Page 148 of 180LVLVLVLVLVLVLVLVLVLVLVLVLVLVLVLVX3LVLVLVLVLVLVLVLVLVLVLVL LD D D D D D D D D D D D D D D D 1 E G H H H H Y G G YV VS S S S S N S S S D S S S S S S X D I S S S S S S SG Y G GI I I I I I I I I I I I I I I I E L Y I I I IS S S S S SF F F R R F Y F R R R R R R R R D L D R RI I I I I I I I IG G G G G G G G H G G G G H G G R D 1H H R R F R Y R R R RE E E E E E E E E E E E E E E E LG G G G G G G K G G G G GD D D D D D D D D D D D DD I 4 E E E E E E E E E E E E EER R R R R R R R RD D D R R L X D D D D D D D D D D D D DCLRL L L L L L L LRLRLRLRLRLRLRL7XLRQQAYRLRLRLRLRLRLRLRLRLRLRLRLRL NERERERERERERERERRREVERAEERERERELY7X02DIRERERERERARER R R R R R RE UL L L L L L L L L L L L L L L L 4 L X V L LE E E E E E EYDYDYDYDVDYDYDYDYDYDYDYDYDYDY Y X Y L A Y YLYLYLYLYLYLYLYLYLYLYLY QR R R R R R R R R RK K R 4 V 4 D G D D D D D D D D D D DE A ASASANAR R R R R R A X 7 3 R R R R R R R R R R R R RS S SASASASASAYAQAKAKAYAYAY1XRA1XXEASASASASASAFASASASASASASAS8E1.4 818.181Mre4.4.4ASrerereN / _sS S SSu_1_2_3EsIne nin naiaiaR E717 7 7 7 7 7 7 7 7 7 7 7 7 7 7 s 8 8 8 8 8 8 8 8 8 8 8 8.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.no momomo 1.1.1.1.1 1 1 1 1 1 1 181S 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 CD D D4 4 4 4.4.4.4.4.4.4.4.4.4:O NDI Q E85950 1 2 3 4 5 6 78686868 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0S 8 868686868686868787878787878787878788888888888888888888898IPTS / 128584488.1 Page 149 of 180LVLVLVLVLVLVLVLVLVLVLVLVLILVLVLVLVLVLVLVLVLVLVL L L L L L L L L LH H G G I G R E G G R F F F F Y R RF V V V V V V V V VS S S S S S S S S S S S S ST T F Q Y V S F Y F H H H Y YI I I I I I I I IS S S S S S S S S S S S S S S S S S SQ V Y Q RI I I I I I I I I I I I I I I I I I I I I I I IG G G G GHGFGFGFGFGRGRGRGNGRGRGRGRGRGRGRGRGAGRGRGRGRGRGRGR R R RE E E E K E E E E E E S E E E E E E V V E E E EG G G GD D D D D D D D D D D D D D D D D D D DE K E E E E E E EE CR R R R R R R R R R R R R RT D D D D D D D D S D D DL L L L L L L L L LR R R R R R R R R R R R R R R R R R RN R R R R R R R R R RLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLR EELELELELELELELELELELELELELELELELELELELELELELELE E E E E A E E E EUYDYDYDYDYDYDYDYDYDYDKDYDYDY Y Y Y Y Y Y Y Y YLYLYLYLYLYLYLYLYLYLY QR R R R R R RD D D D D D D D G D D S D D D D D D D DE A AR R R R R R R H R R R R R R R R R R R R R R R R R RS S SASASASASASASASASASASASASASAFARARASASASASASASASASASAQASASAGAVAVEM A N / SEIR E919.19.19.19.19.19.19 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1 1 1S 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4.4.4.4:O NDI Q E425262728 9 0 1 2 3 4 5 6 7929293938 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6S 9 9 9 939393939393939394949494949494949494959595959595959IPTS / 128584488.1 Page 151 of 180VD11IFVYVYVYVYVYTYVYVYTYVYTYVYVYVYVYTYN V V V I V TS X 1 D S S S S S S S S S S S SY Y Y Y Y Y YI R 1 1 I I I I I I I IS S S S S S S S S S SF L X D 4 Y Y YI I I I I I I I I I I I I I IG 8 RY Y Y Y Y Y Y Y Y Y Y Y Y Y L Y Y Y Y YE X LLLX0GEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEG G G G G G GD 7 8 D 4 D K Q R D N S D D K KE E E E E E EE CR X X N X R R R R RD R K S I D K S T D R RL L 7 L Y L L L L LRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRLRL NRES5XXLQGDIREYERRRERHRERERARHLER R V L R R R R R R R R RE UL I 4 5 E V L L LE A E E S E R E E K E E EYDSI4L L L L L L L L L L L L L L L L L L L L1 DXRX4L8A4YDYDYDYDYDYDYDYQYDYDY Y Y Y Y Y Y Y Y Y F Y YQR R X E A X 1 3 RD D D N D D D D D D D D DER R R R R R R R R R R R R R R R R R R R R RSAFGEGEGR1XRAXYXEASASASAHARASAYASASASASAYASAVASASASASAFASASALASEM A N / StEI ne1L1L2L RlaEvp p pSioBoo oLoLoL 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6:O NDI Q E9 0 1 2 3 4 5 6 7 8 90010203040506070809001112131415161718191S89999999999999999999990101010101010101010101010101010101010101IPTS / 128584488.1 Page 153 of 180

Claims

CLAIMS What is Claimed is:

1. A synthetic TNFR1 binding protein, the binding protein comprising: (a) an amino acid sequence from 30 amino acids to 95 amino acids in length; (b) a net negative charge in phosphate buffered saline (PBS); (c) a binding affinity for TNFR1 stronger than 10 ^M; and (d) a stability profile such that the protein (i) retains at least 90% binding affinity to TNFR1 upon cooling to room temperature after thermal denaturation at 95oC in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to TNFR1 after incubation for 16 hours at 37oC of incubation in PBS relative to the protein under the same conditions prior to incubating; and / or (iii) retains at least 90% binding affinity to TNFR1 in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

2. A synthetic TNFR1 binding protein, the binding protein comprising: (a) an amino acid sequence from 30 amino acids to 95 amino acids in length; (b) a net negative charge in PBS; (c) a binding affinity for TNFR1 stronger than 10 ^M; (d) at least three alpha helices; (e) at least two amino acid loops, where a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., a N-terminal amino acid) of a second alpha helix, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., a C-terminal amino acid) of the second alpha helix to a terminal amino acid (e.g., an N- terminal amino acid) of a third alpha helix; and (f) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one, two or three of the three alpha helices.

3. The synthetic TNFR1 binding protein of claim 1 or 2, wherein binding to TNFR1 occurs through a paratope of the TNFR1 binding protein, which paratope is defined by amino acids X22 - X26 - X31 - X39, wherein X22 is Q, X26 is selected from L, V, I, or A, X31 IPTS / 128584488.1 Page 156 of 180is selected from E or D, and X39 is selected from Y or F, and wherein the amino acid numbering corresponds to the numbering of SEQ ID NO: 993 or 1021.

4. A synthetic TNFR1 binding protein comprising a conformational paratope defined by amino acids X22 - X26 - X31 - X39, wherein X22 is Q, X26 is selected from L, V, I, or A, X31 is selected from E or D, and X39 is selected from Y or F, and wherein the amino acid numbering corresponds to the numbering of SEQ ID NO: 993 or 1021.

5. The synthetic TNFR1 binding protein of claim 3 or 4, wherein X22 is Q, X26 is L, X31 is E, and X39 is Y.

6. The synthetic TNFR1 binding protein of claim 1 or claim 2, comprising a paratope defined by any amino acid combination set forth in Table 2C or Table 8.

7. The synthetic TNFR1 binding protein of any one of claims 1-6, wherein the binding protein comprises one or more of the following features: (a) free of tryptophan amino acids; (b) free of methionine amino acids; (c) free of lysine amino acids; (d) does not comprise an unpaired cysteine amino acid when cysteine amino acids are present in the protein; (e) free of glycosylation sites; (f) free of protease cleavage sites; and (g) soluble up to at least 1 mM in PBS at 4oC for one month.

8. The synthetic TNFR1 binding protein of any one of claims 1-3 and 6-7, wherein the binding affinity is between about 10 ^M to about 0.1 n ^; about 7.5 ^M to about 0.75 nM; about 5 ^M to about 0.5 nM ^ about 2.5 ^M to about 0.25 nM; about 1 ^M to about 1 n ^; about 0.75 ^M to about 1 nM, about 0.5 ^M to about 1 nM; about 0.25 ^M to about 1 n ^ ^ ^about 0.10 ^M to about 1 n ^ ^ ^about 75 n ^ to about 1 n ^ ^ ^about 50 n ^ to about 1 n ^ ^ ^about 25 n ^ to about 1 n ^ ^ ^about 10 n ^ to about 1 n ^ ^ ^and about 5 n ^ to about 1 n ^.

9. The synthetic TNFR1 binding protein of any of claims 1-3 and 6-8, wherein the binding affinity is stronger than about 10 ^M, about 7.5 ^M, about 5 ^M, about 2.5 ^M, about 1 IPTS / 128584488.1 Page 157 of 180^M, about 0.75 ^M, about 0.5 ^M, about 0.25 ^M, about 0.1 ^M, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, and about 0.1 nM.

10. The synthetic TNFR1 binding protein of any one of claims 2-3 or 6-9, wherein the N- terminus of the first alpha helix is preceded by one or more N-terminal amino acids.

11. The synthetic TNFR1 binding protein of claim 10, wherein the N-terminus of the first alpha helix comprises an N-terminal extension.

12. The synthetic TNFR1 binding protein of claim 11, wherein the N-terminal extension has an amino acid sequence comprising that of SEQ ID NO: 1026.

13. The synthetic TNFR1 binding protein of any one of claims 2-3 or 6-10, wherein the C- terminus of the third alpha helix is followed by one or more C-terminal amino acids.

14. The synthetic TNFR1 binding protein of claim 13, wherein the C-terminus of the third alpha helix comprises a C-terminal extension.

15. The synthetic TNFR1 binding protein of claim 14, wherein the C-terminal extension has an amino acid sequence comprising that of SEQ ID NO: 1029.

16. The synthetic TNFR1 binding protein of any one of the preceding claims, wherein the protein comprises from 35 amino acids to 85 amino acids in length, from 35 amino acids to 75 amino acids in length, from 35 amino acids to 65 amino acids in length, from 35 amino acids to 55 amino acids in length, from 35 amino acids to 50 amino acids in length, from 40 amino acids to 85 amino acids in length, from 40 amino acids to 75 amino acids in length, from 40 amino acids to 65 amino acids in length, from 40 amino acids to 55 amino acids in length, or from 40 amino acids to 50 amino acids in length.

17. The synthetic TNFR1 binding protein of claim 16, wherein the protein comprises 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

18. The synthetic TNFR1 binding protein of any one of claims 2-3 or 6-17, wherein the first, second, or third alpha helix, or a combination thereof, is preceded by an alpha helix cap amino acid. IPTS / 128584488.1 Page 158 of 18019. The synthetic TNFR1 binding protein of claim 18, wherein each alpha helix cap amino acid is independently selected from the group consisting of serine, threonine, aspartate, and asparagine.

20. The synthetic TNFR1 binding protein of any one of claims 2-3 and 6-19, wherein (a) the first, second, and / or third alpha helix each contains at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible; (b) the first, second, and / or third alpha helix each contains at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible; (c) the first, second, and / or third alpha helix each contain at least one or two solvent accessible amino acids; (d) the first and / or second loop contains at least one hydrophobic amino acid, or (e) the binding protein comprises any combination of elements selected from (a), (b), (c), and (d).

21. The synthetic TNFR1 binding protein of claim 20, wherein: (a) the second and third alpha helix each contains at least two hydrophobic amino acids; (b) the first, second, and third alpha helix each contains at least one solvent accessible amino acid; (c) the first, second, and third alpha helix each contain at least two hydrophobic and one solvent accessible amino acids; (e) the second and third alpha helix each contains at least four solvent accessible amino acids; and / or (f) the first loop contains at least one hydrophobic amino acid.

22. A multivalent protein comprising a plurality of synthetic TNFR1 binding proteins of any one of claims 1-21.

23. The multivalent protein of claim 22, wherein the multivalent protein is bivalent. IPTS / 128584488.1 Page 159 of 18024. The multivalent protein of claim 22 or 23, wherein the multivalent protein comprises a first synthetic TNFR1 binding protein and a second synthetic TNFR1 binding protein linked together through at least one linker.

25. The multivalent protein of claim 24, wherein a linker connects a C-terminal amino acid of the first synthetic TNFR1 binding protein and an N-terminal amino acid of the second synthetic TNFR1 binding protein.

26. The multivalent protein of claim 25, wherein the linker comprises glycine and serine amino acids (e.g., the amino acid sequence GGS).

27. The multivalent protein of any one of claims 22-26, wherein the multivalent protein has a binding affinity stronger than the binding affinity of each synthetic TNFR1 binding protein alone.

28. A synthetic TNFR1 binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively; and (ii) an amino acid of SEQ ID NO: 1, wherein D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of SEQ ID NO: 3, wherein X4 is E or D; X6 is L or I; X7 is E, R, or Q; X8 is Q, R, or E; and X11 is D or E; (b) D2 comprises an amino acid sequence of SEQ ID NO: 4, wherein X20 is Y or A; X21 is Q, R, E, or D; X24 is L, E, I, V, or T; and X28 is D or E; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 5, wherein X33 is P or E; X34 is Q or E; X37 is R or I; X41 is E, D, N, or R; X43 is Y or H; and X45 is R or E.

29. The synthetic TNFR1 binding protein of claim 28 wherein L1 comprises an amino acid sequence of X12GX14IS and L2 comprises an amino acid sequence of X29GED, wherein X12 is Q, E, R, or N; X14 is L, R, or E; and X29 is R, E, or Q.

30. A synthetic TNFR1 binding protein comprising an amino acid sequence of SEQ ID NO: 1, wherein X4 is E or D; X6 is L or I; X7 is E, R, or Q; X8 is Q, R, or E; X11 is D or E; X12 is Q, E, R, or N; X14 is L, R, or E; X20 is Y, or A; X21 is Q, R, E, or D; X24 is L, IPTS / 128584488.1 Page 160 of 180E, I, V, or T; X28 is D or E; X29 is R, E, or Q; X33 is P or E; X34 is Q or E; X37 is R or I; X41 is E, D, N, or R; X43 is Y or H; and X45 is R or E.

31. A synthetic TNFR1 binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 1 or 6-17.

32. A synthetic TNFR1 binding protein comprising: an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively, and wherein D1, D2, and D3 independently comprise any of the following combinations: D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11 wherein X2 is A or G; X3 is R, A, D, Q, Y, K, H, S, L, N, T, G, or V; X4 is D, A, E, K, S, I, N, V, T, L, Q, F, R, H, Y, or G; X5 is Y, T, G, I, Q, R, A, H, V, S, E, F, K, or D; X7 is E, L, A, R, Q, I, D, K, F, G, V, T, H, N, S, or Y; X8 is R, Y, E, L, V, A, F, Q, N, K, H, T, or G; X10 is R, A, T, V, E, or Y; and X11 is R, D, E, K, S, L, Q, T, F, I, V, G, H, or N; D2 comprises an amino acid sequence of X17X18LX20X21QLX24X25X26X27X28, wherein X17 is D, F, Y, G, H, T, R, A, K, S, H, N, E, V, Q, or I; X18 is V, I, T, Y, F, or D; X20 is E, Y, Q, H, K, N, X, R, V, T, A, S, F, or L; X21 is Q, Y, D, G, H, I, E, T, V, N, F, R, S, or A; X24 is I, S, N, V, G, T, Q, E, D, L, R, F, Y, or A; X25 is D, S, Y, E, Q, or T; X26 is L, I, or V; X27 is L, I, or V; and X28 is N, L, D, R, T, I, Y, A, E, Q, G, V, F, or H; and D3 comprises an amino acid sequence set forth in SEQ ID NO: 21, wherein X33 is E, G, S, A, D, Y, F, Q, I, R, H, K, or N; X34 is N, Q, H, R, Y, V, I, D, A, L, F, K, S, E, or G; X37 is I, V, T, E, R, A, K, or S; X41 is N, Y, L, I, H, D, F, Q, V, S, R, E, A, or T; X43 is Y or F; X44 is I or L; and X45 is E, D, T, R, F, L, S, A, I, Y, V, or N.

33. The synthetic TNFR1 binding protein of claim 32, wherein L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E, Q, S, F, I, K, T, R, L, Y, A, D, N, G, V, or H; X13 is G, R, H, K, S, L, Q, N, I, or Y; X14 is R, Y, G, A, L, I, H, F, V, K, T, E, Q, S, or N; X16 is S, N, G, D, T, IPTS / 128584488.1 Page 161 of 180or E; X29 is R, I, Q, N, E, S, K, V, L; and X32 is D, F, Y, Q, E, R, N, L, H, A, I, S, V, G, T, or K.

34. A synthetic TNFR1 binding protein comprising: an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein, D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively, and D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of AX3X4X5LX7X8X9RX11, wherein X3 is R, A, D, Q, Y, or K; X4 is D, A, E, K, S, or I; X5 is Y, T, or G; X7 is E, L A, R, Q, I, D, K, or F; X8 is R or Y; X9 is L or Y; and X11 is R, D, E, K, S or L; D2 comprises an amino acid sequence of SEQ ID NO: 24, wherein X17 is D, F, Y, G, or H; X20 is E, Y, Q, H, or K; X21 is Q, Y, D, G, or H; X24 is I, S, N, V, or G; X25 is D, S, or Y; X27 is L or I; and X28 is N, L, D or R; and D3 comprises an amino acid sequence of SEQ ID NO: 25, wherein X33 is E, G, or S; X34 is N, Q, or H; X41 is N, Y, L, or I; X43 is Y or F; and X45 is E, D, T or R; (b) D1 comprises an amino acid sequence of SEQ ID NO: 50, wherein X4 is D or E; and X7 is E or K; D2 comprises an amino acid sequence of SEQ ID NO: 51, wherein X17 is Y, G, or D; and X20 is K, Q, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 52, wherein X33 is E or S; X34 is Q or R; and X41 is N, H, or D; (c) D1 comprises an amino acid sequence of SEQ ID NO: 75, wherein X3 is R, H, S, or K; X4 is D, N, V, T, S, E, L, Q, F, K, or I; X5 is I, Y, Q, or R; X7 is E, L, G, A, K, F, V, Q, or R; X8 is R, Y, E, L, V, or A; and X11 is D, K, Q, or T; D2 comprises an amino acid sequence of SEQ ID NO: 76, wherein X17 is D, G, Y, or H; X18 is V, I, or T; X20 is Y, K, R, V, T, E, Q, or A; X21 is Q, G, D, or H; X24 is I, T, or G; X25 is D, E, or Q; and X28 is D, L, T, I, Y, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 77, wherein X33 is A, S, D, E, Y, or F; X34 is Q, Y, V, I, or D; X41 is Y, N, or D; X43 is Y or F; and X45 is E, D, F, or L; IPTS / 128584488.1 Page 162 of 180(d) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11, wherein X2 is A or G; X3 is R, L, or A; X4 is D, E, K, or R; X5 is A or Y; X7 is E, G, R, L, K, or V; X8 is R, F, Y, or Q; X10 is R or A; and X11 is D or Q; D2 comprises an amino acid sequence of SEQ ID NO: 150, wherein X17 is F, H, T, D, R, A, K, S, or Y; X21 is Q or I; X24 is Q, I, E, or V; X28 is D or N; and D3 comprises an amino acid sequence of SEQ ID NO: 151, wherein X33 is E, G, Y, Q, I, A, F, S, or R; X37 is I or V; X41 is N, F, Y, or L; and X43 is Y or F; (e) D1 comprises an amino acid sequence of SEQ ID NO: 194, wherein X3 is R, D, A, H, N, Q or T; X4 is D, R, N, E, or Q; X7 is E, K, Q, G, R, L, or I; X8 is R, E, A, N, or Q; and X11 is D, F, I, or K; D2 comprises an amino acid sequence of SEQ ID NO: 195, wherein X17 is S, F, Y, H, A, G, R, N, E, D, or T; X18 is V, Y, F, D, or T; X20 is Y, Q, or R; X21 is Q, Y, E, H, or T; X24 is S, I, V, E, or N; X25 is D or E; X26 is L or V; X27 is L or I; and X28 is D, A, or L; and D3 comprises an amino acid sequence of SEQ ID NO: 196, wherein X33 is E, D, F, G, or Y; X34 is Q, I, A, V, N, D, or L; X37 is I or T; X43 is Y or F; and X45 is E, F, or S; (f) D1 comprises an amino acid sequence of AX3X4X5LX7X8LX10X11, wherein X3 is R, Y, A, S, Q, or G; X4 is Q, E, F, D, K, R, or Y; X5 is Y, T, or A; X7 is E, R, G, T, L, H, F, K, A, I, or V; X8 is R, K, or Y; X10 is R or T; and X11 is D or V; D2 comprises an amino acid sequence of SEQ ID NO: 274, wherein X17 is F, D, Y, H, K, S, R, V, A, G, or I; X18 is V or T; X20 is Y, R, E, V, K, H, or T; X21 is Q, Y, V, H, G, or N; X24 is D, I, G, N, or L; X25 is D or E; X26 is L, I, or V; X27 is L or V; and X28 is E, L, D, Q, T, G, or I; and D3 comprises an amino acid sequence of SEQ ID NO: 275, wherein X33 is E, S, D, G, A, H, Y, K, or N; X34 is Q, Y, F, N, R, H, I, K, or Z; X37 is I, E, R, or A; X41 is Y, F, N, L, or I; X43 is Y or F; X44 is I or L; and X45 is E or A; (g) D1 comprises an amino acid sequence of SEQ ID NO: 346, wherein X4 is D, F, or H; and X7 is K, E, G, or R; D2 comprises an amino acid sequence of SEQ ID NO: 347, wherein X17 is Y, H, V, or F; X20 is E, K, or Y; X24 is I or S; X26 is L or V; and X27 is L or V; and D3 comprises an amino acid sequence of SEQ ID NO: 348, wherein X34 is F, N, Y, or H; X37 is I or R; X41 is Y or N; and X45 is E, D, or A; IPTS / 128584488.1 Page 163 of 180(h) D1 comprises an amino acid sequence of SEQ ID NO: 368, wherein X3 is R or H; X4 is D, Y, N, I, or H; X5 is Y or H; X7 is E, A, or K; X8 is R, A, or H; X11 is D, K, or R; D2 comprises an amino acid sequence of SEQ ID NO: 369, wherein X17 is A, G, Q, D, Y, T, N, F, R, K, H, or I; and D3 comprises an amino acid sequence of SEQ ID NO: 370, wherein X34 is H, N, Y, E, G, D, R, F, A, or I; and X41 is L, D, N, Y, or F; (i) D1 comprises an amino acid sequence of SEQ ID NO: 405, wherein X3 is R or Q; X4 is D, Q, V, or S; X5 is Y, T, or V; X7 is E, R, or G; X8 is R or N; and X11 is D, F, or G; D2 comprises an amino acid sequence of SEQ ID NO: 406, wherein X17 is G, Y, A, T, R, F, S, I, Q, or H; X18 is Y or V; X20 is Y, K, E, or T; and X21 is Q or F; and D3 comprises an amino acid sequence of SEQ ID NO: 407, wherein X34 is F, I, D, S, H, R, N, or Y; and X41 is Q, V, Y, N, S, L, I, F, D, R, E, or H; (j) D1 comprises an amino acid sequence of AX3X4X5LX7X8LX10X11, wherein X3 is R, Q, K, A, or E; X4 is D, H, G, S, K, I, L, E, T, or V; X5 is Y, S, E, V, or F; X7 is E, R, G, I, L, or K; X8 is R, T, or G; X10 is R or V; and X11 is D, F, or H; D2 comprises an amino acid sequence of SEQ ID NO: 447, wherein X17 is H, Y, S, G, R, K, A, T, F, or N; X18 is V, D, or I; X20 is Y or Q; X21 is G, Y, Q, H, or F; X24 is I, G, L, V, S, R, or D; X25 is Q, D, or E; X26 is L, I, or V; X27 is L, I, or V; and X28 is D, A, L, R, E, T, V, F, G, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 448, wherein X33 is E, H, N, S, A, or G; X34 is Q, E, R, or V; X37 is I, A, E, or V; X41 is V, Q, F, Y, N, I, L, D, or E; X43 is Y or F; and X45 is E, A, S, L, I, F, or Y; (k) D1 comprises an amino acid sequence of SEQ ID NO: 524, wherein X4 is D, I, N, H, L, K, or A; X5 is K, E, or Y; X8 is R, H, or V; and X11 is D or I; D2 comprises an amino acid sequence of SEQ ID NO: 525, wherein X17 is R, F, N, K, Y, G, H, S, T, or A; X20 is Y, E, N, Q, K, S, or H; X21 is Q, N, E, G, or R; X24 is I or G; and X26 is L or V; and D3 comprises an amino acid sequence of SEQ ID NO: 526, wherein X34 is Q or F; and X41 is Y, F, I, L, D, V, A, E, S, N, or T; (l) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LRX11, wherein X2 is A or G; X3 is R or Q; X4 is D, S, F, L, T, K, or A; X5 is Y or F; X7 is E, G, T, V, or R; X8 is R or L; and X11 is D or V; D2 comprises an amino acid sequence of SEQ ID NO: 565, wherein X17 is D or Q; X18 is V or I; X20 is Y, A, E, Q, T, or R; X21 is Q or G; IPTS / 128584488.1 Page 164 of 180X24 is I, F, G, or Y; and X28 is D, H, or N; and D3 comprises an amino acid sequence of SEQ ID NO: 566, wherein X33 is E, S, G, Y, or D; and X34 is Q or E; X37 is I, A, or V; X41 is F, D, V, Y, I, or L; X43 is Y or F; and X45 is E, Y, I, V, or F; (m) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11, wherein X2 is A or G; X3 is R, Y, S, or A; X4 is E, D, Y, T, A, L, G, F, K, N, or R; X5 is Y, A, I, V, E, G, or S; X7 is E, Q, R, N, A, S, K, or T; X8 is R, G, F, L, Q, K, A, or N; X10 is T, R, V, or E; and X11 is D, L, H, Q, I, N, or F; D2 comprises an amino acid sequence of SEQ ID NO: 605, wherein X17 is H, Q, D, Y, F, T, G, or K; X20 is Y, H, S, K, R, A, E, V, Q, F, L, or T; X21 is Q, G, H, or E; X24 is I, A, or R; X25 is D, T, or Q; and X28 is D, R, I, or F; and D3 comprises an amino acid sequence of SEQ ID NO: 606, wherein X33 is E, S, or G; X34 is L, R, Q, S, or H; X37 is I, A, K, S, or V; X41 is Y, I, L, F, V, D, A, H, or T; X43 is Y or F; X44 is I or L; X45 is E, I, N, Y, or L; (n) D1 comprises an amino acid sequence of SEQ ID NO: 677, wherein X3 is R, Q, V, or G; X4 is D, G, F, R, V, Q, E, L, Y, A, or T; X5 is Y, A, I, V, G, E, or H; X7 is K, E, Y, Q, A, S, L; X8 is R, F, Q, T, K, or Y; and X11 is D, L, R, F, N, Q, K, or V; D2 comprises an amino acid sequence of SEQ ID NO: 678, wherein X17 is F, Q, D, G, or Y; X18 is V, D, or T; X20 is Y, H, Q, R, K, A, T, V, L, or E; and X21 is Q, G, H, or Y; and D3 comprises an amino acid sequence of SEQ ID NO: 679, wherein X34 is K, Y, L, H, F, N, A, R, G, D, E, or I; X37 is I or A; and X41 is V, N, I, F, Y, L, E, or H; (o) D1 comprises an amino acid sequence of SEQ ID NO: 739, wherein X3 is R, V, or H; X4 is D, E, H, G, or K; X5 is Y, D, G, V, or S; X7 is E or R; and X8 is R, F, or V; D2 comprises an amino acid sequence of SEQ ID NO: 740, wherein X18 is V or I; X20 is T, Y, F, K, or V; X21 is Q, R, S, or H; and X25 is Q or D; and D3 comprises an amino acid sequence of SEQ ID NO: 741, wherein X34 is Y, S, G, R, D, F, N, V, A, L, H, I, or K; and X41 is N, F, L, I, Y, V, or D; (p) D1 comprises an amino acid sequence of SEQ ID NO: 787, wherein X4 is D or I; X5 is Y or T; and X8 is R or K; D2 comprises an amino acid sequence of SEQ ID NO: 788, wherein X1 is G, H, D, or Y; X20 is K or Y; and X21 is Q or Y; and D3 comprises an amino acid sequence of SEQ ID NO: 789, wherein X34 is Q, Y, L, N, or G; and X41 is N, F, I, Y, L, V, or D; IPTS / 128584488.1 Page 165 of 180(q) D1 comprises an amino acid sequence of X2X3X4X5LX7X8LX10X11, wherein X2 is A or G; X3 is R, H, or Y; X4 is D, Y, F, L, or K; X5 is Y, I, S, A, L, T, or V; X7 is E, Y, I, L, K, S, R, G, A, or Q; X8 is R, L, T, Y, V, or E; X10 is R or Y; and X11 is D, H, or R; D2 comprises an amino acid sequence of SEQ ID NO: 813, wherein X18 is T or V; X20 is Y, Q, K, R, T, S, N, A, or F; and X21 is Q, A, G, H, E, or S; and D3 comprises an amino acid sequence of SEQ ID NO: 814, wherein X41 is V, F, D, Y, I, S, E, or L; (r) D1 comprises an amino acid sequence of SEQ ID NO: 875, wherein X4 is G or D; and X7 is E or A; D2 comprises an amino acid sequence of SEQ ID NO: 876, wherein X17 is H, Y, or G; and X20 is Y, K, or R; and D3 comprises an amino acid sequence of SEQ ID NO: 877, wherein X34 is Q, N, or E; and X41 is N, L, Y, F, D, or I; and (s) D1 comprises an amino acid sequence of SEQ ID NO: 897, wherein X2 is A or G; X3 is R, Y, K, or H; X4 is D, S, L, or G; X5 is Y, T, or K; X7 is R, K, I, E, G, or A; and X11 is D, T, or S; D2 comprises an amino acid sequence of SEQ ID NO: 898, wherein X17 is D, H, Y, F, A, Q, T, R, G, I, E, V, or S; X18 is V, I, or F; X20 is Y, K, T, or S; or X21 is Q, A, Y, or G; and D3 comprises an amino acid sequence of SEQ ID NO: 899, wherein X41 is N, L, D, Y, T, V, K, I, A, E, or F.

35. The synthetic TNFR1 binding protein of claim 34, wherein (a) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E, Q, S, or F; X13 is G or R; X14 is R, Y, G, A, or L; X16 is S, N, G, or D; X29 is R, I, Q, N, E, S, or K; and X32 is D, F, or Y; (b) L1 comprises an amino acid sequence of SEQ ID NO: 990; and L2 comprises an amino acid sequence of X29GEX32, wherein X29 is R, E, or S; and X32 is Q, E, Y, or R; (c) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, I, K, T, F, R, S, L, Q, Y, or A; X13 is G, H, K, S, or L; X14 is R, L, I, H, Y, F, A, V, or K; X16 is S, D, or T; and X32 is R, F, Q, Y, N, D, L, H, or A; IPTS / 128584488.1 Page 166 of 180(d) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, D, I, T, R, N, Y, or K; X13 is G or R; X14 is R or K; and X32 is Y, I, R, or L; (e) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E, N, F, G, S, Y, or V; X13 is G, H, R, K, or Q; X14 is F, R, K, H, I, T, Y, E, or Q; X29 is R, K, or N; and X32 is L, A, R, N, Q, S, Y, F, or V; (f) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of X29GED, wherein X12 is E, F, R, G, Y, L, or H; X13 is G, K, N, H, or R; X14 is R, F, H, Q, or Y; and X29 is R, V, K, or L; (g) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, N, F, D, I, or K; X13 is G, R, or K; X14 is R, E, F, or K; and X32 is D or A; (h) L1 comprises an amino acid sequence of SEQ ID NO: 991; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X14 is R, A, Y, K, or L; (i) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, T, S, or D; X13 is G, R, or K; and X14 is R, L, or S; (j) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E, T, H, V, K, N, R, L, or F; X13 is G, N, R, H, or K; X14 is R, V, E, Q, H, K, Y, or F; and X32 is D, Q, or R; (k) L1 comprises an amino acid sequence of X12X13RIS; and L2 comprises an amino acid sequence of X29GED, wherein X12 is E, G, S, or K; X13 is G or R; and X29 is R or L; (l) L1 comprises an amino acid sequence of X12X13X14IS; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, L, Q, G, D, Y; X13 is G, H, or I; X14 is Y, F, L, H, V, or I; IPTS / 128584488.1 Page 167 of 180(m) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, K, T, R, I, G, Y, or V; X13 is G, Q, R, K, or H; X14 is R, Y, or K; and X16 is S or E; (n) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, D, F, T, R, S, N, Y, G, L, Q, or K; X13 is G, K, or Y; X14 is H or R; and X16 is S, T, or D; (o) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of SEQ ID NO: 992, wherein X12 is E, Y, S, G, N, I, L; X13 is G, K, or N; X14 is F, Y, A, G, Q, L, I, V, H, K; and X16 is S or D; (p) L1 comprises an amino acid sequence of EX13X14IS; and L2 comprises an amino acid sequence of RGEX32, wherein X13 is G, H, or R; X14 is R or Y; and X32 is D, G, S, F, or Y; (q) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, L, A, Q, I, H, F, or Y; X13 is G, R, H; X14 is Q, R, F, Y, H, or S; X16 is S, N, or D; and X32 is Y, N, V, F, R, L, S, T, Q, or I; (r) L1 comprises an amino acid sequence of EX13X14IS; and L2 comprises an amino acid sequence of RGEX32, wherein X13 is G or K; X14 is R, H, F, or Y; X32 is Y, R, F, or D; and (s) L1 comprises an amino acid sequence of X12X13X14IX16; and L2 comprises an amino acid sequence of RGEX32, wherein X12 is E, K, S, or V; X13 is H, G, or R; X14 is R, H, I, Q, V, Y, F, N, or A; X16 is S or D; and X32 is F, N, Y, A, S, L, Q, R, I, E, K.

36. A synthetic TNFR1 binding protein comprising: an amino acid sequence of SEQ ID NO: 18, wherein X1 is A, S, D, E, F, G, H, I, K, L, N, Q, R, S, V, or Y; X2 is A or G; X3 is R, A, D, Q, Y, K, H, S, L, N, T, G, or V; X4 is D, A, E, K, S, I, N, V, T, L, Q, F, R, H, Y, or G; X5 is Y, T, G, I, Q, R, A, H, V, S, E, F, K, or D; X7 is E, L, A, R, Q, I, D, K, F, G, V, T, H, N, S, or Y; X8 is R, Y, E, L, V, A, F, Q, N, K, H, T, or G; X10 is R, A, T, V, E, or Y; X11 is R, D, E, K, S, L, Q, T, F, I, V, G, H, or N; X12 is E, Q, S, F, I, K, T, R, L, Y, A, D, N, G, V, or H; X13 is G, R, H, K, S, L, Q, IPTS / 128584488.1 Page 168 of 180N, I, or Y; X14 is R, Y, G, A, L, I, H, F, V, K, T, E, Q, S, or N; X16 is S, N, G, D, T, or E; X17 is D, F, Y, G, H, T, R, A, K, S, H, N, E, V, Q, or I; X18 is V, I, T, Y, F, or D; X20 is E, Y, Q, H, K, N, X, R, V, T, A, S, F, or L; X21 is Q, Y, D, G, H, I, E, T, V, N, F, R, S, or A; X24 is I, S, N, V, G, T, Q, E, D, L, R, F, Y, or A; X25 is D, S, Y, E, Q, or T; X26 is L, I, or V; X27 is L, I, or V; X28 is N, L, D, R, T, I, Y, A, E, Q, G, V, F, or H; X29 is R, I, Q, N, E, S, K, V, or L; X32 is D, F, Y, Q, E, R, N, L, H, A, I, S, V, G, T, or K; X33 is E, G, S, A, D, Y, F, Q, I, R, H, K, or N; X34 is N, Q, H, R, Y, V, I, D, A, L, F, K, S, E, or G; X37 is I, V, T, E, R, A, K, or S; X41 is N, Y, L, I, H, D, F, Q, V, S, R, E, A, or T; X43 is Y or F; X44 is I or L; X45 is E, D, T, R, F, L, S, A, I, Y, V, or N; and X46 is S, G, R, D, T, E, L, I, F, V, or A; X47 is R, V, I, L, T, Y, E, S, K, G, Q, D, N, A, or H.

37. A synthetic TNFR1 binding protein comprising: (a) an amino acid sequence of SEQ ID NO: 22, wherein X1 is F, S, R, D, or L; X3 is R, A, D, Q, Y, or K; X4 is D, A, E, K, S, or I; X5 is Y, T, or G; X7 is E, L, A, R, Q, I, D, K, or F; X8 is R or Y; X9 is L or Y; X11 is R, D, E, K, S, or L; X12 is E, Q, S, or F; X13 is G or R; X14 is R, Y, G, A, or L; X16 is S, N, G, or D; X17 is D, F, Y, G, or H; X20 is E, Y, Q, H, or K; X21 is Q, Y, D, G, or H; X24 is I, S, N, V, or G; X25 is D, S, or Y; X27 is L, or I; X28 is N, L, D, or R; X29 is R, I, Q, N, E, S, or K; X32 is D, F, or Y; X33 is E, G, or S; X34 is N, Q, or H; X41 is N, Y, L, or I; X43 is Y or F; X45 is E, D, T, or R; X46 is S, G, or R; and X47 is R, V, I, L, T, Y, E, or S; (b) an amino acid sequence of SEQ ID NO: 49, wherein X1 is S, F, V, or R; X4 is D or E; X7 is E or K; X17 is Y, G, D; X20 is K, Q, or N; X29 is R, E, or S; X32 is Q, E, Y, or R; X33 is E or S; X34 is Q or R; X41 is N, H, or D; X46 is S, D, or R; and X47 is K, R, E, L, G, Q, or I; (c) an amino acid sequence of SEQ ID NO: 74, wherein X1 is L, S, Y, F, G, T, I, V, K, H, D, Q, or R; X3 is R, H, S, or K; X4 is D, N, V, T, S, E, L, Q, F, K, or I; X5 is I, Y, Q, or R; X7 is E, L, G, A, K, F, V, Q, or R; X8 is R, Y, E, L, V, or A; X11 is D, K, Q, or T; X12 is E, I, K, T, F, R, S, L, Q, Y, or A; X13 is G, H, K, S, or L; X14 is R, L, I, H, Y, F, A, V, or K; X16 is S, D, or T; X17 is D, G, Y, H; X18 is V, I, or T; X20 is Y, K, R, V, T, E, Q, or A; X21 is Q, G, D, or H; X24 is I, T, or G; X25 is D, E, or Q; X28 is D, L, T, I, Y, or N; X32 is R, F, Q, Y, N, D, L, H, or A; X33 is A, S, D, E, Y, or F; X34 is Q, Y, V, IPTS / 128584488.1 Page 169 of 180I, D; X41 is Y, N, or D; X43 is Y or F; X45 is E, D, F, or L; X46 is S, D, T, E, or L; and X47 is R, T, L, Q, I, D, S, Y, or N; (d) an amino acid sequence of SEQ ID NO: 148, wherein X1 is S or V; X2 is A or G; X3 is R, L, or A; X4 D, E, K, or R; X5 is A or Y; X7 is E, G, R, L, K, or V; X8 is R, F, Y, or Q; X10 is R or A; X11 is D or Q; X12 is E, D, I, T, R, N, Y, or K; X13 is G or R; X14 is R or K; X17 is F, H, T, D, R, A, K, S, or Y; X21 is Q or I; X24 is Q, I, E, or V; X28 is D or N; X32 is Y, I, R, or L; X33 is E, G, Y, Q, I, A, F, S, or R; X37 is I or V; X41 is N, F, Y, or L; X43 is Y or F; X46 is S or G; X47 is R, T, E, Q, L, N, Y, I, or F; (e) an amino acid sequence of SEQ ID NO: 193, wherein X1 is S, V, Y, F, K, R, G, or A; X3 is R, D, A, H, N, Q, or T; X4 is D, R, N, E, or Q; X7 is E, K, Q, G, R, L, or I; X8 is R, E, A, N, or Q; X11 is D, F, I, or K; X12 is E, N, F, G, S, Y, or V; X13 is G, H, R, K, or Q; X14 is F, R, K, H, I, T, Y, E, or Q; X17 is S, F, Y, H, A, G, R, N, E, D, or T; X18 is V, Y, F, D, or T; X20 is Y, Q, or R; X21 is Q, Y, E, H, or T; X24 is S, I, V, E, or N; X25 is D or E; X26 is L or V; X27 is L or I; X28 is D, A, or L; X29 is R, K, or N; X32 is L, A, R, N, Q, S, Y, F, or V; X33 is E, D, F, G, or Y; X34 Q, I, A, V, N, D, or L; X37 is I or T; X43 is Y or F; X45 is E, F, or S; X46 is S, I, E, F, V, A, or D; and X47 is R, A, E, T, L, I, N, G, Q, or S; (f) an amino acid sequence of SEQ ID NO: 272, wherein X1 is S, R, K, F, or L; X3 is R, Y, A, S, Q, or G; X4 is Q, E, F, D, K, R, or Y; X5 is Y, T, or A; X7 is E, R, G, T, L, H, F, K, A, I, or V; X8 is R, K, or Y; X10 is R or T; X11 is D or V; X12 is E, F, R, G, Y, L, or H; X13 is G, K, N, H, or R; X14 is R, F, H, Q, or Y; X16 is S or D; X17 F, D, Y, H, K, S, R, V, A, G, or I; X18 is V or T; X20 is Y, R, E, V, K, H, or T; X21 is Q, Y, V, H, G, or N; X24 is D, I, G, N, or L; X25 is D or E; X26 is L, I, or V; X27 is L or V; X28 is E, L, D, Q, T, G, or I; X29 is R, V, K, or L; X33 is E, S, D, G, A, H, Y, K, or N; X34 is Q, Y, F, N, R, H, I, K, or A; X37 is I, E, R, or A; X41 is Y, F, N, L, or I; X43 is Y or F; X44 is I or L; X45 is E or A; X46 is S, L, D, I, E, or A; X47 is R, V, L, T, I, G, Q, E, F, S, or Y; (g) an amino acid sequence of SEQ ID NO: 345, wherein X4 is D, F, or H; X7 is K, E, G, or R; X12 is E, N, F, D, I, or K; X13 is G, R, or K; X14 is R, E, F, or K; X17 is Y, H, V, or F; X20 is E, K, or Y; X24 is I or S; X26 is L or V; X27 is L or V; X32 is D or A; X34 IPTS / 128584488.1 Page 170 of 180is F, N, Y, or H; X37 is I or R; X41 is Y or N; X45 is E, D, or A; X46 is S, L, or F; X47 is R, L, V, Q, E, N, or I; (h) an amino acid sequence of SEQ ID NO: 367, wherein X1 is S, F, or V; X3 is R or H; X4 is D, Y, N, I, or H; X5 is Y or H; X7 is E, A, or K; X8 is R, A, or H; X11 is D, K, or R; X14 is R, A, Y, K, or L; X17 is A, G, Q, D, Y, T, N, F, R, K, H, or I; X18 is V, D, F, or T; X20 is H, K, V, or Y; X21 is Q, H, or Y; X34 is H, N, Y, E, G, D, R, F, A, or I; X41 is L, D, N, Y, or F; and X47 is R or I; (i) an amino acid sequence of SEQ ID NO: 404, wherein X1 is S, I, or Y; X3 is R or Q; X4 is D, Q, V, is S; X5 is Y, T, or V; X7 is E, R, or G; X8 is R or N; X11 is D, F, or G; X12 is E, T, S, or D; X13 is G, R, or K; X14 is R, L, or S; X17 is G, Y, A, T, R, F, S, I, Q, or H; X18 is Y or V; X20 is Y, K, E, or T; X21 is Q or F; X34 is F, I, D, S, H, R, N, or Y; X41 is Q, V, Y, N, S, L, I, F, D, R, E, or H; (j) an amino acid sequence of SEQ ID NO: 445, wherein X1 is S, Q, F, R, or K; X3 is R, Q, K, A, or E; X4 is D, H, G, S, K, I, L, E, T, V; X5 is Y, S, E, V, or F; X7 is E, R, G, I, L, or K; X8 is R, T, or G; X10 is R or V; X11 is D, F, or H; X12 is E, T, H, V, K, N, R, L, or F; X13 is G, N, R, H, or K; X14 is R, V, E, Q, H, K, Y, or F; X17 is H, Y, S, G, R, K, A, T, F, or N; X18 is V, D, or I; X20 is Y or Q; X21 is G, Y, Q, H, or F; X24 is I, G, L, V, S, R, or D; X25 is Q, D, or E; X26 is L, I, or V; X27 is L, I, or V; X28 is D, A, L, R, E, T, V, F, G, or N; X29 is R or K; X32 is D, Q, or R; X33 is E, H, N, S, A, or G; X34 is Q, E, R, or V; X37 is I, A, E, or V; X41 is V, Q, F, Y, N, I, L, D, or E; X43 is Y or F; X45 is E, A, S, L, I, F, or Y; X46 is S, L, or E; and X47 is R or Y; (k) an amino acid sequence of SEQ ID NO: 523, wherein X1 is S or K; X4 is D, I, N, H, L, K, or A; X5 is K, E, or Y; X8 is R, H, or V; X11 is D or I; X12 is E, G, S, or K; X13 is G or R; X17 is R, F, N, K, Y, G, H, S, T, or A; X20 is Y, E, N, Q, K, S, or H; X21 is Q, N, E, G, or R; X24 is I or G; X26 is L or V; X29 is R or L; X34 is Q or F; and X41 is Y, F, I, L, D, V, A, E, S, N, or T; (l) an amino acid sequence of SEQ ID NO: 563, wherein X1 is V, S, or L; X2 is A or G; X3 is R or Q; X4 is D, S, F, L, T, K, or A; X5 is Y or F; X7 is E, G, T, V, or R; X8 is R or L; X11 is D or V; X12 is E, L, Q, G, D, or Y; X13 is G, H, or I; X14 is Y, F, L, H, V, or I; X17 is D or Q; X18 is V or I; X20 is Y, A, E, Q, T, or R; X21 is Q or G; X24 is I, F, G, IPTS / 128584488.1 Page 171 of 180or Y; X28 is D, H, or N; X33 is E, S, G, Y, or D; X34 is Q or E; X37 is I, A, or V; X41 is F, D, V, Y, I, or L; X43 is Y or F; X45 is E, Y, I, V, or F; X46 is S, D, E, L, or A; and X47 is S, V, R, E, or L; (m) an amino acid sequence of SEQ ID NO: 603, wherein X1 is S, R, K, I, Q, L, Y, or F; X2 is A or G; X3 is R, Y, S, or A; X4 is E, D, Y, T, A, L, G, F, K, N, or R; X5 is Y, A, I, V, E, G, or S; X7 is E, Q, R, N, A, S, K, or T; X8 is R, G, F, L, Q, K, A, or N; X10 is T, R, V, or E; X11 is D, L, H, Q, I, N, or F; X12 is E, K, T, R, I, G, Y, or V; X13 is G, Q, R, K, or H; X14 is R, Y, or K; X16 is S or E; X17 is H, Q, D, Y, F, T, G, or K; X20 is Y, H, S, K, R, A, E, V, Q, F, L, or T; X21 is Q, G, H, or E; X24 is I, A, or R; X25 is D, T, or Q; X28 is D, R, I, or F; X33 is E, S, or G; X34 is L, R, Q, S, or H; X37 is I, A, K, S, or V; X41 is Y, I, L, F, V, D, A, H, or T; X43 is Y or F; X44 is I or L; X45 is E, I, N, Y, or L; X46 is S, E, D, or L; and X47 is R, H, E, I, A, V, L, S, T, Q, or K; (n) an amino acid sequence of SEQ ID NO: 676, wherein X1 is S, G, K, V, Q, or E; X3 is R, Q, V, or G; X4 is D, G, F, R, V, Q, E, L, Y, A, or T; X5 is Y, A, I, V, G, E, or H; X7 is K, E, Y, Q, A, S, or L; X8 is R, F, Q, T, K, or Y; X11 is D, L, R, F, N, Q, K, or V; X12 is E, D, F, T, R, S, N, Y, G, L, Q, or K; X13 is G, K, or Y; X14 is H or R; X16 is S, T, or D; X17 is F, Q, D, G, or Y; X18 is V, D, or T; X20 is Y, H, Q, R, K, A, T, V, L, or E; X21 is Q, G, H, or Y; X34 is K, Y, L, H, F, N, A, R, G, D, E, or I; X37 is I or A; and X41 is V, N, I, F, Y, L, E, or H; (o) an amino acid sequence of SEQ ID NO: 738, wherein X1 is S, F, Q, R, or I; X3 is R, V, or H; X4 is D, E, H, G, or K; X5 is Y, D, G, V, or S; X7 is E or R; X8 is R, F, or V; X12 is E, Y, S, G, N, I, or L; X13 G, K, or N; X14 is F, Y, A, G, Q, L, I, V, H, or K; X16 is S or D; X18 is V or I; X20 is T, Y, F, K, or V; X21 is Q, R, S, or H; X25 is Q or D; X34 is Y, S, G, R, D, F, N, V, A, L, H, I, or K; X41 is N, F, L, I, Y, V, or D; and X47 is R or E; (p) an amino acid sequence of SEQ ID NO: 786, wherein X1 is S, K, or Y; X4 is D or I; X5 is Y or T; X8 is R or K; X13 is G, H, or R; X14 is R or Y; X17 is G, H, D, or Y; X20 is K or Y; X21 is Q or Y; X32 is D, G, S, F, or Y; X34 is Q, Y, L, N, or G; X41 is N, F, I, Y, L, V, or D; and X47 is R, I, or T; IPTS / 128584488.1 Page 172 of 180(q) an amino acid sequence of SEQ ID NO: 811, wherein X1 is S, H, G, L, N, Y, Q, or K; X2 is A or G; X3 is R, H, or Y; X4 is D, Y, F, L, or K; X5 is Y, I, S, A, L, T, or V; X7 is E, Y, I, L, K, S, R, G, A, or Q; X8 is R, L, T, Y, V, or E; X10 is R or Y; X11 is D, H, or R; X12 is E, L, A, Q, I, H, F, or Y; X13 is G, R, or H; X14 is Q, R, F, Y, H, or S; X16 is S, N, or D; X18 is T or V; X20 is Y, Q, K, R, T, S, N, A, or F; X21 is Q, A, G, H, E, or S; X32 is Y, N, V, F, R, L, S, T, Q, or I; and X41 V, F, D, Y, I, S, E, or L; (r) an amino acid sequence of SEQ ID NO: 874, wherein X1 is S or F; X4 is G or D; X7 is E or A; X13 is G or K; X14 is R, H, F, or Y; X17 is H, Y, or G; X20 is Y, K, or R; X32 is Y, R, F, or D; X34 is Q, N, or E; X41 is N, L, Y, F, D, or I; and X47 is R, E; and (s) an amino acid sequence of SEQ ID NO: 896, wherein X1 is S, F, R, Q, G, V; X2 is A or G; X3 is R, Y, K, or H; X4 is D, S, L, or G; X5 is Y, T, or K; X7 is R, K, I, E, G, or A; X11 is D, T, or S; X12 is E, K, S, or V; X13 is H, G, or R; X14 is R, H, I, Q, V, Y, F, N, or A; X16 is S or D; X17 is D, H, Y, F, A, Q, T, R, G, I, E, V, or S; X18 is V, I, or F; X20 is Y, K, T, or S; X21 is Q, A, Y, or G; X32 is F, N, Y, A, S, L, Q, R, I, E, or K; X41 is N, L, D, Y, T, V, K, I, A, E, or F; and X47 is R or E.

38. A synthetic TNFR1 binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 26-48, 53-73, 78-147, 152-192, 197-271, 276-344, 349-366, 371-403, 408-444, 449-522, 527-562, 567-602, 607-675, 680-737, 742-785, 790-810, 815-873, 878-895, and 900-957.

39. A synthetic TNFR1 binding protein comprising: an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively, and D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of SEQ ID NO: 959, wherein X5 is Y or T; X6 is L or I; X7 is R, Q, E, D, or K; X8 is R or Y; X9 is L or Y; and X11 is D or E; (b) D2 comprises an amino acid sequence of SEQ ID NO: 960, wherein X17 is D, G, or H; X20 is E, K, or Y; X21 is G or Q; X23 is L or Y; and X24 is N or I; and IPTS / 128584488.1 Page 173 of 180(c) D3 comprises an amino acid sequence of SEQ ID NO: 961, wherein X33 is E or S; and X43 is S or Y.

40. The synthetic TNFR1 binding protein of claim 39, wherein: (i) L1 comprises an amino acid sequence of X12GX14IS; and (ii) L2 comprises an amino acid sequence of X29GEX32, wherein X12 is E or Q; X14 is Y or R; X29 is R, I, E, Q, N, or S; and X32 is D or Y.

41. A synthetic TNFR1 binding protein comprising an amino acid sequence of SEQ ID NO: 958, wherein X5 is Y or T; X6 is L or I; X7 is R, Q, E, D, or K; X8 is R or Y; X9 is L or Y; X11 is D or E; X12 is E or Q; X14 is Y or R; X17 is D, G, or H; X20 is E, K, or Y; X21 G or Q; X24 is N or I; X29 is R, I, E, Q, N, or S; X32 is D or Y; X33 is E or S; X43 is F or Y; and X47 is R, Y, E, or L.

42. A synthetic TNFR1 binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 878 and 962-982.

43. A synthetic TNFR1 binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively; and (ii) an amino acid of SEQ ID NO: 993, wherein D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of SEQ ID NO: 994, wherein X4 is D, Q, or N; X5 is Y or F; X7 is E, R, H, A, S, or K; X8 is R, Y, L, or V; and X11 is D, K, Q, R, N, S, I, or T; (b) D2 comprises an amino acid sequence of SEQ ID NO: 995, wherein X18 is V, T, N, or I; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 996, wherein X34 is Q, D, E, or N; X40 is A or V; and X41 is L or D.

44. The synthetic TNFR1 binding protein of claim 43, wherein L1 comprises an amino acid sequence of X12GX14IS and L2 comprises an amino acid sequence of X29GED, wherein X12 is E or A; X14 is Y or L; and X29 is R, I, or L. IPTS / 128584488.1 Page 174 of 18045. A synthetic TNFR1 binding protein comprising an amino acid sequence of SEQ ID NO: 993, wherein X1 is S, H, R, Y, V, F, or L; X4 is D, Q, or N; X5 is Y or F; X7 is E, R, H, A, S, or K; X8 is R, Y, L, or V; X11 is D, K, Q, R, N, S, I, or T; X12 is E or A, or N; X14 is Y or L; X18 is V, T, N, or I; X29 is R, I, or L; X34 is Q, D, E, or N; X40 is A or V; X41 is L or D; and X47 is R, E, L, Q, or T.

46. A synthetic TNFR1 binding protein comprising an amino acid sequence selected from any of SEQ ID NOs: 997-1020.

47. A synthetic TNFR1 binding protein comprising: (i) an amino acid sequence arranged in a primary structure of D1-L1-D2-L2-D3 (Formula I), wherein D1, D2, and D3 are domains 1, 2, and 3, respectively, and L1 and L2 are loops 1 and 2, respectively; and (ii) an amino acid of SEQ ID NO: 1021, wherein D1, D2, and D3 independently comprise any of the following combinations: (a) D1 comprises an amino acid sequence of SEQ ID NO: 1022, wherein X8 is R or Y; and X11 is D, K, or R; (b) D2 comprises an amino acid sequence of SEQ ID NO: 1023; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 1024, wherein X41 is L or D.

48. The synthetic TNFR1 binding protein of claim 47, wherein L1 comprises an amino acid sequence of SEQ ID NO: 1025 and L2 comprises an amino acid sequence of X29GED, wherein X12 is E or A; and X29 is R or I.

49. A synthetic TNFR1 binding protein comprising an amino acid sequence of SEQ ID NO: 1021, wherein X8 R or Y; X11 is D, K, or R; X12 is E or A; X29 is R or I; X41 is L or D; and X47 is R or E.

50. The synthetic TNFR1 binding protein of any one of claims 28-49 wherein the binding protein has a binding affinity for TNFR1 stronger than 10 ^M.

51. The synthetic TNFR1 binding protein of any one of claims 28-50, wherein the binding affinity is between about 10 ^M to about 0.1 n ^; about 7.5 ^M to about 0.75 nM; about 5 ^M to about 0.5 nM ^ about 2.5 ^M to about 0.25 nM; about 1 ^M to about 1 n ^; about IPTS / 128584488.1 Page 175 of 1800.5 ^M to about 1 nM; about 0.25 ^M to about 1 n ^ ^ ^about 0.10 ^M to about 1 n ^ ^ ^about 75 n ^ to about 1 n ^ ^ ^about 50 n ^ to about 1 n ^ ^ ^about 25 n ^ to about 1 n ^ ^ ^about 10 n ^ to about 1 n ^ ^ ^and about 5 n ^ to about 1 n ^.

52. The synthetic TNFR1 binding protein of any one of claims 28-51, wherein the binding affinity is stronger than about 10 ^M, about 7.5 ^M, about 5 ^M, about 2.5 ^M, about 1 ^M, about 0.75 ^M, about 0.5 ^M, about 0.25 ^M, about 0.1 ^M, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, and about 0.1 nM.

53. The synthetic TNFR1 binding protein of any one of claims 28, 29, 32, 33, 34, 35, 39, 40, 43, 44, 47, or 48, wherein D1 is flanked by one or more N-terminal amino acids and / or D3 is flanked by one or more C-terminal amino acids.

54. The synthetic TNFR1 binding protein of claim 53, wherein the N-terminus of the first alpha helix comprises an N-terminal extension.

55. The synthetic TNFR1 binding protein of claim 54, wherein the N-terminal extension has an amino acid sequence comprising that of SEQ ID NO: 1026.

56. The synthetic TNFR1 binding protein of any one of claims 53-55, wherein the C-terminus of the third alpha helix comprises a C-terminal extension.

57. The synthetic TNFR1 binding protein of claim 56, wherein the C-terminal extension has an amino acid sequence comprising that of SEQ ID NO: 1029.

58. The synthetic TNFR1 binding protein of any one of claims 28-31, wherein the amino acid sequence of the binding protein has at least 65 (e.g., 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5) percent identity to that of SEQ ID NO:

1.

59. The synthetic TNFR1 binding protein of claim any one of claims 32-38, wherein the amino acid sequence of the binding protein has at least 85 (e.g., 90, 95, 96, 97, 98, 99, 99.5) percent identity to that of SEQ ID NO:

9. IPTS / 128584488.1 Page 176 of 18060. The synthetic TNFR1 binding protein of any one of claims 39-42, wherein the amino acid sequence of the binding protein has at least 70 (e.g., 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5) percent identity to that of SEQ ID NO:

30.

61. The synthetic TNFR1 binding protein of any one of claims 43-46, wherein the amino acid sequence of the binding protein has at least 80 (e.g., 85, 90, 95, 96, 97, 98, 99, 99.5) percent identity to that of SEQ ID NO:

966.

62. The synthetic TNFR1 binding protein of any one of claims 43-46, wherein the amino acid sequence of the binding protein has at least 85 (e.g., 90, 95, 96, 97, 98, 99, 99.5) percent identity to that of SEQ ID NO:

997.

63. The synthetic TNFR1 binding protein of any one of claims 1-62, wherein the protein has an amino acid sequence comprising or according to one or more sequences set forth in Table 12.

64. A multivalent protein comprising a plurality of synthetic TNFR1 binding proteins of any one of claims 28-62.

65. The multivalent protein of claim 64, wherein the multivalent protein is bivalent.

66. The multivalent protein of claim 64 or 65, wherein the multivalent protein comprises a first synthetic TNFR1 binding protein and a second synthetic TNFR1 binding protein linked together through at least one linker.

67. The multivalent protein of claim 66, wherein a linker connects a C-terminal amino acid of the first synthetic TNFR1 binding protein and the N-terminal amino acid of the second synthetic TNFR1 binding protein.

68. The multivalent protein of claim 67, wherein the linker comprises glycine and serine amino acids (e.g., the amino acid sequence GGS).

69. The multivalent protein of any one of claims 64-68, wherein the multivalent protein has a binding affinity stronger than the binding affinity of each synthetic TNFR1 binding protein alone. IPTS / 128584488.1 Page 177 of 18070. The multivalent protein of any one of claims 64-69, wherein the multivalent protein comprises a synthetic TNFR1 binding protein having an amino acid sequence, wherein the amino acid sequence comprises an amino acid sequence set forth in Table 12.

71. The multivalent protein of any one of claims 64-70, wherein the multivalent protein has an amino acid sequence, wherein the amino acid sequence comprises an amino acid sequence set forth in Table 12.

72. A pharmaceutical composition comprising the synthetic TNFR1 binding protein of any one of claims 1-21 or 28-61, or the multivalent protein of any one of claims 22-27 or 62- 71; and a pharmaceutically acceptable carrier.

73. The pharmaceutical composition of claim 72, wherein the synthetic TNFR1 binding protein or the multivalent protein further comprises an effector molecule.

74. A method of targeting TNFR1, the method comprising contacting a cell that expresses TNFR1 on its cell surface with a composition comprising the synthetic TNFR1 binding protein of any one of claims 1-21 or 28-61, or the multivalent protein of any one of claims 22-27 or 62-71.

75. A method of modulating TNFR1 activity, the method comprising contacting a cell that expresses TNFR1 on its cell surface with a composition comprising the synthetic TNFR1 binding protein any one of claims 1-21 or 28-61, or the multivalent protein of any one of claims 22-27 or 62-71.

76. The method of claim 74 or 75, wherein the synthetic TNFR1 binding protein or the multivalent protein further comprises an effector molecule.

77. The method of any one of claims 74-76, wherein the synthetic TNFR1 binding protein or the multivalent protein inhibits or reduces TNFR1 activity in the presence of a TNFR1 ligand (e.g., TNF ^) relative to TNFR1 activity in the presence of the TNFR1 ligand (e.g., TNF ^) but in the absence of the synthetic TNFR1 binding protein or the multivalent protein.

78. A method of decreasing TNFR1-mediated activity in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 72 or 73. IPTS / 128584488.1 Page 178 of 18079. A method of treating inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 72 or 73.

80. The method of claim 78 or 79, wherein the subject is diagnosed as having rheumatoid arthritis, juvenile idiopathic arthritis, plaque psoriasis including pediatric plaque psoriasis, psoriatic arthritis, axial spondylitis including ankylosing spondylitis and non-radiographic axial spondyloarthritis, Crohn’s disease, ulcerative colitis including pediatric ulcerative colitis, uveitis, or Hidradenitis Suppurativa. IPTS / 128584488.1 Page 179 of 180

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