TNF-alpha binding agents and methods of use thereof

Abstract

Provided are D-peptide binding agents of TNF [alpha], multimers thereof, and methods of using D-peptides and multimers thereof.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 483839, filed February 8, 2023, the disclosure of which is incorporated herein by reference in its entirety. Declaration of sequence list

[0002] The sequence list XML related to this application is provided in XML format and is incorporated herein by reference. The XML file containing the sequence list is named 4298-P1WO_Seq_List_20240207.xml. This XML file is 689,475 bytes long, created on February 7, 2024, and is submitted electronically to the Patent Center along with this specification. Government License Rights Statement

[0003] This invention was developed with government support from the National Institutes of Health (NIH) SBIR grant program (1R43DK117777-01). The government holds certain rights to this invention. background

[0004] FDA-approved anti-TNFα therapies include four antibodies: (adalimumab) (Infliximab), (Certuzumab) and SIMPONI (golimumab), and a soluble form of TNFR, (Etanercept). These drugs are used to treat a wide range of inflammatory conditions, including ulcerative colitis, Crohn's disease, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and psoriasis. Given... Anti-TNFα is the world's best-selling drug, and its value in treating chronic inflammatory diseases is evident. However, approved anti-TNFα biologics are limited by factors such as: the need for parenteral administration; systemic distribution associated with immunosuppression and increased risk of infection; and immunogenicity associated with anti-drug antibodies (ADAs), which leads to loss of therapeutic activity. This invention addresses this need, as well as other needs. Brief

[0005] This section aims to introduce some concepts in a simplified form, which will be further described in the "Detailed Explanation" section below. This section is not intended to identify the key features of the claimed subject matter, nor is it intended to assist in determining the scope of the claimed subject matter.

[0006] Provided herein are D-peptide-based inhibitors of TNFa. In some embodiments, the peptides inhibit TNFa activity by binding to TNFa. In some embodiments, the peptides inhibit TNFa activity by binding to TNFa and blocking its binding to its receptors, TNFR1 and / or TNFR2. The TNFi peptides comprise a core TNFa binding domain composed of D-amino acids.

[0007] In some embodiments, provided is a D-peptide or salt thereof, wherein the peptide comprises a core TNFa binding domain of D-amino acids and has the amino acid sequence, C-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -C(SEQ ID NO: 111), wherein each of X 2 to X 11 is a D-amino acid; and X 2 is the D form of any typical L-amino acid except cysteine; X 3 is the D form of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), or Leu (L); X 4 is a polar amino acid including Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gin (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G); X 5 is the D form of any typical L-amino acid except cysteine; X 6 is the D form of any typical L-amino acid except cysteine; X 7 is the D form of Phe (F); X 8 is the D form of Asn (N), X 9 is the D form of Asn (N), X 10 is the D form of Trp (W) or Tyr (Y); X 11 is the D form of Trp (W), Gin (Q), Tyr, or His (H); and C represents the D form of cysteine.

[0008] In some embodiments, provided is a D-peptide or salt thereof, wherein the peptide comprises a core TNFa binding domain of D-amino acids and has the amino acid sequence, C*-X 2 -X 3 -X 4 -X 5 -X6 -X 7 -X 8 -X 9 -X 10 -X 11 -C*(SEQ ID NO: 112), wherein X 2 each of X 11 is a D-amino acid; and X 2 is the D form of any typical L-amino acid except cysteine; X 3 is the D form of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), and Leu (L); X 4 is a polar amino acid including Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gin (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), and Gly (G); X 5 is the D form of any typical L-amino acid except cysteine; X 6 is the D form of any typical L-amino acid except cysteine; X 7 is the D form of Phe (F); X 8 is the D form of Asn (N), X 9 is the D form of Asn (N), X 10 is the D form of Trp (W) and Tyr (Y); X 11 is the D form of Trp (W), Tyr (Y), Gin (Q), or His (H); C represents the D form of cysteine; and * represents an optional intramolecular bond.

[0009] In some embodiments, a D-peptide or salt thereof is provided, wherein the peptide comprises a core TNFα binding domain of D-amino acids and has the amino acid sequence, C*-X 2 -[W / F / Y]-[Polar]-X 5 -X 6 -F-N-N-[W / Y]-W-C*(SEQ ID NO: 113), wherein X 2 is the D form of any typical L-amino acid except Cys, X 5 is the D form of any typical L-amino acid except cysteine, X 6 is the D form of any typical L-amino acid except cysteine, Polar represents a D-amino acid including one of R, K, H, E, D, Q, N, T, S, P, A, or G, and * represents an optional intramolecular disulfide bond.

[0010] In some embodiments, a D-peptide or a salt thereof is provided, wherein the peptide comprises a core TNFα-binding domain of a D-amino acid and has the following amino acid sequence, CX 2 -X 3 -X 4 -X 5 -X 6 -FFNX 10 -X 11 -C(SEQ ID NO: 1), where X 2 To X 6 X 10 Each of them is a D-amino acid or its D-α-amino acid analogue; and aX 2 It is any one of D-type Thr(T), Val(V), His(H), Leu(L), Gln(Q), Ala(A), Ile(I), Met(M) or Trp(W), or its D-α-amino acid analogue; bX 3 It is D-type Trp(W), Phe(F), Tyr(Y), Ser(S), His(H) or Leu(L), or their D-α-amino acid analogs; cX 4 They are polar amino acids, including D-type Arg(R), Lys(K), His(H), Glu(E), Asp(D), Gln(Q), Asn(N), Thr(T), Ser(S), Pro(P), Ala(A) or Gly(G), or their D-α-amino acid analogs; dX 5 It is D-type Pro, Trp, His, Gln, Gly, Arg, Ala, Val, or Leu, or their D-α-amino acids; eX 6 It is D-type Arg(R), His(H), Lys(K), Glu(E), Gln(Q), Val(V), Leu(L), Ser(S) or Ala(A), or their D-α-amino acid analogs; fX 10 It is D-type Trp(W) or Tyr(Y), or their D-α-amino acid analogs; gX 11 It is a D-type Trp(W), Tyr(Y), Gln(Q), or His(H) or its D-α-amino acid analogue; and hC represents D-cysteine, F represents D-phenylalanine, and N represents D-asparagine, or a D-α-amino acid analog of C, F, or N.

[0011] In some embodiments, the TNFi peptide comprises a core TNFα binding domain of D-amino acids and has the following amino acid sequence: C*-X 2 -X 3 -X 4 -X 5 -X 6 -F-N-N-X 10 -X 11 -C*(SEQ ID NO: 2), wherein each of X 1 to X 6 , X 10 , and X 11 is a D-amino acid; and a. X 2 is a D-Thr (T), Val (V), His (H), Leu (L), Gin (Q), Ala (A), He (I), Met (M), or Trp (W), or a D-a-amino acid analog thereof; b. X 3 is a D-Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), or Leu (L), or a D-a-amino acid analog thereof; c. X 4 is a polar amino acid, including D-Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gin (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G), or a D-a-amino acid analog thereof; d. X 5 is a D-Pro (P), Trp (W), His (H), Gin (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), or a D-a-amino acid analog thereof; e. X 6 is a D-Arg (R), His (H), Lys (K), Glu (E), Gin (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-a-amino acid analog thereof; f. X 10 is a D-Trp (W) or Tyr (Y), or a D-a-amino acid analog thereof; and g. X 11 is a D-Trp (W), Tyr (Y), Gin (Q), or His (H); C denotes a D-Cysteine; wherein F denotes a D-Phenylalanine; N denotes a D-Asparagine; and * denotes an optional intramolecular disulfide bond.

[0012] In some embodiments, provided are D-peptides or salts thereof, wherein the core TNFα binding domain has the following amino acid sequence: C-X 2 -[W / F / Y]-X 4 -X 5 -X 6 -F-N-N-[W / Y]-W-C (SEQ ID NO: 3), wherein X 2 is D-Thr (T), Val (V), His (H), Leu (L), Gin (Q), Ala (A), He (I), Met (M), or Trp (W); X 4 is D-Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gin (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G); X 5 is D-Pro (P), Trp (W), His (H), Gin (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), X 6 is D-Arg (R), His (H), Lys (K), Glu (E), Gin (Q), Val (V), Leu (L), Ser (S), or Ala (A); and * indicates an optional intramolecular disulfide bond between the indicated cysteine residues.

[0013] In some embodiments, provided are D-peptides or salts thereof, further comprising an intramolecular disulfide bond between cysteine residues of the core TNFα binding domain.

[0014] In some embodiments, provided are D-peptides or salts thereof having the amino acid sequence set forth in any one of SEQ ID NOs: 1-3, wherein: a. X 2 is D-Thr, Val, His, Leu, Gin, Ala, He, Met, or Trp, or a D-α-amino acid analog thereof; b. X 2 is D-Thr, Val, His, Leu, or Gin, or a D-α-amino acid analog thereof; c. X 2 is D-Thr, Val, His, or Leu, or a D-α-amino acid analog thereof; d. X 2 is D-Thr, Val, or His, or a D-α-amino acid analog thereof; e. X 2 is D-Thr or Val, or a D-α-amino acid analog thereof; f. X2 is D-Thr, or a D-a-amino acid analog thereof; or g.X 2 is D-Val, or a D-a-amino acid analog thereof.

[0015] In some embodiments, there is provided a D-peptide having the amino acid sequence set forth in any one of SEQ ID NOs: 1 or 2, or a salt thereof, wherein: a.X 3 is D-Trp, Phe, Tyr, or Ser, or a D-a-amino acid analog thereof; b.X 3 is D-Trp, Phe, or Tyr, or a D-a-amino acid analog thereof; c.X 3 is D-Trp or Phe, or a D-a-amino acid analog thereof; d.X 3 is D-Trp, or a D-a-amino acid analog thereof; or e.X 3 is D-Phe, or a D-a-amino acid analog thereof.

[0016] In some embodiments, there is provided a D-peptide having the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or a salt thereof, wherein: a.X 4 is D-Arg, His, Gin, Asn, Lys, Thr, or Ser, or a D-a-amino acid analog thereof; b.X 4 is D-Arg, His, Gin, or Asn, or a D-a-amino acid analog thereof; c.X 4 is D-Arg, His, or Gin, or a D-a-amino acid analog thereof; d.X 4 is D-Arg, Gin, or Asn, or a D-a-amino acid analog thereof; or e.X 4 is D-Gln, or a D-a-amino acid analog thereof.

[0017] In some embodiments, there is provided a D-peptide having the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or a salt thereof, wherein: a.X 5 is D-Pro, Trp, His, Gin, Gly, Arg, Ala, Val, or Leu, or a D-a-amino acid analog thereof; b.X 5is D-form Pro, Trp, His, Gin, Gly, Arg, or Val, or a D-a-amino acid analog thereof; c. X 5 is D-form Pro, Trp, or His, or a D-a-amino acid analog thereof; d. X 5 is D-form Pro or Trp, or a D-a-amino acid analog thereof; or e. X 5 is D-form Pro, or a D-a-amino acid analog thereof.

[0018] In some embodiments, there is provided a D-peptide having the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or a salt thereof, wherein: a. X 6 is D-form Arg, His, Lys, Glu, Gin, Val, Leu, Ser, or Ala, or a D-a-amino acid analog thereof; b. X 6 is D-form Arg, His, Lys, Glu, Gin, Val, or Leu, or a D-a-amino acid analog thereof; c. X 6 is D-form Arg, His, Lys, Glu, or Gin, or a D-a-amino acid analog thereof; d. X 6 is D-form Arg, His, Lys, or Glu, or a D-a-amino acid analog thereof; e. X 6 is D-form Arg, His, or Lys, or a D-a-amino acid analog thereof; f. X 6 is D-form Arg, or a D-a-amino acid analog thereof; g. X 6 is D-form Lys, or a D-a-amino acid analog thereof, or h. X 6 is D-form His, or a D-a-amino acid analog thereof.

[0019] In some embodiments, there is provided a D-peptide having the amino acid sequence set forth in any one of SEQ ID NOs: 1 or 2, or a salt thereof, wherein: a. X 10 is D-form Trp, or a D-a-amino acid analog thereof; or b. X 10 is D-form Tyr, or a D-a-amino acid analog thereof.

[0020] In some embodiments, provided are D-peptides having the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or salts thereof, wherein: a. X 11 is D-Trp (W), Tyr (Y), or Gin (Q), or a D-alpha amino acid analog thereof; b. X 11 is D-Tyr (Y), or c. X 11 is D-Trp (W), or a D-alpha amino acid analog thereof.

[0021] In some embodiments, provided are D-peptides, or salts thereof, wherein each of X 1 to X 6 , X 10 , and X 11 is a D-amino acid. C is D-cysteine. F is D-phenylalanine, and N is D- asparagine.

[0022] In some embodiments, provided are D-peptides, or salts thereof, wherein the core TNF alpha binding domain has an amino acid sequence selected from the group consisting of: a. CVWQPKFNNYWC (SEQ ID NO: 4); b. CVWQPRFNNYWC (SEQ ID NO: 5); c. CTFQPRFNNYWC (SEQ ID NO: 6); d. CTFQPRFNNWWC (SEQ ID NO: 7); e. CSFQPRFNNYWC (SEQ ID NO: 8); f. CSFQPRFNNWWC (SEQ ID NO: 9); g. CVFQPRFNNYWC (SEQ ID NO: 10); h. CVFQPRFNNWWC (SEQ ID NO: 11); i. CTFQWRFNNYWC (SEQ ID NO: 12); j. CLYQPVFNNWWC (SEQ ID NO: 13); k. CVFQAAFNNYWC (SEQ ID NO: 14); l. CVFQHHFNNWWC (SEQ ID NO: 15); m. CHFNPRFNNWWC (SEQ ID NO: 16); n. CVWQPHFNNYWC (SEQ ID NO: 17); o. CVFQGRFNNWWC (SEQ ID NO: 18); p. CVFQHRFNNWWC (SEQ ID NO: 19); q. CVFNPRFNNWWC (SEQ ID NO: 20); r. CVFKPRFNNWWC (SEQ ID NO: 21); s. CAYQRQFNNWWC (SEQ ID NO: 22); t. CWFEHRFNNWHC (SEQ ID NO: 23); u. CHFQHRFNNWWC (SEQ ID NO: 24); v. CHFQPRFNNWWC (SEQ ID NO: 25); w. CTYQPRFNNWWC (SEQ ID NO: 26); x. CQFQPRFNNWQC (SEQ ID NO: 27); or y. CHFSQRFNNWWC (SEQ ID NO: 28).

[0023] In some embodiments, D-peptides or salts thereof are provided, wherein the core TNFα binding domain comprises an amino acid sequence set forth in SEQ ID NOs: 77-110.

[0024] In some embodiments, D-peptides or salts thereof are provided, further comprising a tag sequence linked to the N-terminus of the peptide. In some embodiments, D-peptides or salts thereof are provided, wherein the tag comprises the amino acid sequence D-Asp or D-Asp D-Asp (DD). In some embodiments, D-peptides or salts thereof are provided, further comprising a tag sequence linked to the C-terminus of the peptide. In some embodiments, D-peptides or salts thereof are provided, wherein the tag comprises the amino acid sequence D-GGEEEK (SEQ ID NO: 30) or D-GGRRRK (SEQ ID NO: 31), wherein each amino acid residue is a D-amino acid.

[0025] In some embodiments, D-peptides or salts thereof are provided, wherein the N-terminus of the peptide comprises a cap. In some embodiments, D-peptides or salts thereof are provided, wherein the cap comprises an acetyl group or a protecting group. In some embodiments, D-peptides or salts thereof are provided, wherein the C-terminus of the peptide comprises a cap. In some embodiments, D-peptides or salts thereof are provided, wherein the cap comprises an amide group or a protecting group.

[0026] In some embodiments, provided is a D-peptide or salt thereof, wherein the peptide comprises a cap and a tag, each as described herein.

[0027] In some embodiments, provided is a D-peptide or salt thereof, further comprising a polyethylene glycol (PEG) group. In some embodiments, provided is a D-peptide or salt thereof, further comprising a linker. In some embodiments, provided is a D-peptide or salt thereof, wherein the linker comprises a PEG group. In some embodiments, provided is a D-peptide or salt thereof, wherein the PEG group is attached to the N-terminus of the D-peptide. In some embodiments, provided is a D-peptide or salt thereof, wherein the PEG group is attached to the C-terminus of the D-peptide. In some embodiments, provided is a D-peptide or salt thereof, wherein each PEG group is selected from a PEG group having 1 to 48 subunits, 1 to 30 subunits, 1 to 24 subunits, or 1 to 12 subunits. In some embodiments, provided is a D-peptide or salt thereof, wherein each PEG group is selected from a PEG group having 6 subunits, 8 subunits, 10 subunits, or 12 subunits.

[0028] In some embodiments, provided is a multimer of any D-peptide described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, provided is a multimer of any D-peptide described herein, or a salt thereof, wherein the multimer is a dimer. In some embodiments, provided is a multimer of any D-peptide described herein, or a salt thereof, wherein the multimer is a trimer. In some embodiments, provided is a multimer of any D-peptide described herein, or a salt thereof, further comprising a multimeric backbone attached to the D-peptide, optionally through a linker. In some embodiments, provided is a multimer of any D-peptide described herein, or a salt thereof, wherein the multimeric backbone is trimeric. In some embodiments, provided is a multimer of any D-peptide described herein, or a salt thereof, wherein the multimeric backbone is tetrameric.

[0029] In some embodiments, there is provided a multimer of any of the D-peptides described herein, or a salt thereof, wherein the tri-functional crosslinker is tris(succinimidyl) aminotriacetate (TSAT), tris-succinimidyl (6-aminohexanoyl) aminotriacetate (LC-TSAT), Fmoc backbone (bis(2,5-dioxopyrrolidin-1-yl) 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)heptanedioate, Fmoc backbone with PEG27 chains, cyclohex backbone (tris(2,5-dioxopyrrolidin-1-yl)cyclohexane-1,3,5-tricarboxylate), nitro backbone (bis(2,5-dioxopyrrolidin-1-yl) 4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-4-nitroheptanedioate), amine backbone (bis(2,5-dioxopyrrolidin-1-yl) 4-amino-4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)heptanedioate), amine-PEG27 backbone, cholesteryl backbone, isotetrameric PEG backbone based on 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propanoic acid backbone, multimeric backbone based on 4-amino-4-(2-carboxyethyl)heptanedioic acid or 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propanoic acid.

[0030] In some embodiments, there is provided a multimer of any of the D-peptides described herein, or a salt thereof, wherein the multimer has a structure selected from: a. Fmoc-[peptide-PEG12-K-amide]3; b. Fmoc-[peptide-PEG4-K-amide]3; c. Fmoc-[peptide-PEG8-K-amide]3; d. Fmoc-[Ac-K-PEG12-peptide-amide]3; e. [peptide-PEG6-K-amide]3-Fmoc; f. [peptide-PEG12-K-amide]3-PEG27-Fmoc; g. [peptide-PEG12-K-amide]3-Fmoc; h. [peptide-PEG12-K-amide]3-cyclohex; i. [peptide-PEG12-K-amide]3-nitro; j. [peptide-PEG12-K-amide]3-PEG27-amine; k. [peptide-PEG12-K-amide]3-amine; l. [Peptide-PEG12-K-amide]3-PEG27-biotin; and m. [Peptide-PEG12-K-amide]3-PEG27-cholesterol; and wherein the peptide is a D-peptide.

[0031] In some embodiments, there is provided a D-peptide or salt thereof having an amino acid sequence selected from at least one of the following: a. DDCVWQPKFNNYWC (SEQ ID NO: 32); b. DGACVWQPKFNNYWC (SEQ ID NO: 38); c. DDCVWQPRFNNYWC (SEQ ID NO: 39); d. DDCVWQPRFNNYWCGGRRRK (SEQ ID NO: 40); e. DCVWQPKFNNYWC (SEQ ID NO: 56); f. DDDCVWQPRFNNYWC (SEQ ID NO: 60); g. DDCTFQPRFNNWWC (SEQ ID NO: 61); h. DDCVFQPRFNNYWC (SEQ ID NO: 62); i. DDCSFQPRFNNYWC (SEQ ID NO: 63); and j. DDCVFQPRFNNWWC (SEQ ID NO: 64).

[0032] In some embodiments, there is provided a multimer of any of the D-peptides described herein or salt thereof, comprising a D-peptide having an amino acid sequence selected from at least one of the following: a. DDCVWQPKFNNYWC (SEQ ID NO: 32); b. DGACVWQPKFNNYWC (SEQ ID NO: 38); c. DDCVWQPRFNNYWC (SEQ ID NO: 39); d. DDCVWQPRFNNYWCGGRRRK (SEQ ID NO: 40); e. DCVWQPKFNNYWC (SEQ ID NO: 56); f. DDDCVWQPRFNNYWC (SEQ ID NO: 60); g. DDCTFQPRFNNWWC (SEQ ID NO: 61); h. DDCVFQPRFNNYWC (SEQ ID NO: 62); i. DDCSFQPRFNNYWC (SEQ ID NO: 63); and j. DDCVFQPRFNNWWC (SEQ ID NO: 64).

[0033] In some embodiments, provided are pharmaceutical compositions comprising at least one D-peptide or multimer thereof described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier. In some embodiments, provided are pharmaceutical compositions formulated for parenteral administration. In some embodiments, provided are pharmaceutical compositions formulated for intravenous, intramuscular, or subcutaneous administration. In some embodiments, provided are pharmaceutical compositions formulated for oral administration. In some embodiments, provided are pharmaceutical compositions formulated for topical administration. In some embodiments, provided are pharmaceutical compositions formulated for topical administration to the skin (dermis) or the eye. In some embodiments, provided are pharmaceutical compositions formulated for rectal administration.

[0034] In some embodiments, provided are lyophilized compositions comprising at least one D-peptide or multimer described herein, or a salt or pharmaceutically acceptable salt thereof, and a stabilizer. In some embodiments, provided are lyophilized compositions of any of the pharmaceutical compositions described herein and a stabilizer. In some embodiments, provided are rehydrated solutions of any of the lyophilized compositions described herein.

[0035] In some embodiments, provided are methods of treating a TNFα-mediated disease, the method comprising administering an effective amount of any of the D-peptides or salts or pharmaceutically acceptable salts thereof described herein, or any of the multimers or salts thereof described herein, or any of the pharmaceutical compositions described herein. In some embodiments, the TNFα-mediated disease is adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, cutaneous lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (polyarticular), hidradenitis suppurativa, uveitis (intermediate, posterior, or pan-uveitis), or radiographic negative axial spondyloarthritis.

[0036] In some embodiments, the TNFa-mediated disease is an inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is adult Crohn’s disease, pediatric Crohn’s disease, or ulcerative colitis. In some embodiments, the D-peptide or pharmaceutically acceptable salt thereof described herein, or the multimer or pharmaceutically acceptable salt thereof described herein, or the pharmaceutical composition described herein is administered orally. In some embodiments, the D-peptide or pharmaceutically acceptable salt thereof described herein, or the multimer or pharmaceutically acceptable salt thereof described herein, or the pharmaceutical composition described herein is administered rectally. In some embodiments, the D-peptide or pharmaceutically acceptable salt thereof described herein, or the multimer or pharmaceutically acceptable salt thereof described herein, or the pharmaceutical composition described herein is administered parenterally.

[0037] In some embodiments, the TNFa-mediated disease is an inflammatory skin disease. In some embodiments, the inflammatory skin disease is selected from plaque psoriasis and cutaneous lupus. In some embodiments, the D-peptide or pharmaceutically acceptable salt thereof described herein, or the multimer or pharmaceutically acceptable salt thereof described herein, or the pharmaceutical composition described herein is administered topically. In some embodiments, the D-peptide or pharmaceutically acceptable salt thereof described herein, or the multimer or pharmaceutically acceptable salt thereof described herein, or the pharmaceutical composition described herein is administered parenterally. In some embodiments, the D-peptide or pharmaceutically acceptable salt thereof described herein, or the multimer or pharmaceutically acceptable salt thereof described herein, or the pharmaceutical composition described herein is administered orally.

[0038] In some embodiments, the TNFa-mediated disease is an inflammatory disease, such as systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, idiopathic arthritis (polyarticular), or radiographic negative axial spondyloarthritis. In some embodiments, the D-peptide or pharmaceutically acceptable salt thereof described herein, or the multimer or pharmaceutically acceptable salt thereof described herein, or the pharmaceutical composition described herein is administered parenterally.

[0039] In any of the embodiments described herein, the parenteral administration can be intravenous, subcutaneous, and intramuscular administration.

[0040] In some embodiments, there is provided a method of reducing TNFα-mediated inflammation, comprising administering to a subject any D-peptide described herein, or a pharmaceutically acceptable salt thereof, or any multimer described herein, or a pharmaceutically acceptable salt thereof, or any pharmaceutical composition described herein. In some embodiments, there is provided a method of inhibiting TNFα, comprising administering to a subject any D-peptide described herein, or a pharmaceutically acceptable salt thereof, or any multimer described herein, or a pharmaceutically acceptable salt thereof, or any pharmaceutical composition described herein. In some embodiments, there is provided a method of reducing a TNFα-mediated inflammatory response, comprising administering to a subject any D-peptide described herein, or a pharmaceutically acceptable salt thereof, or any multimer described herein, or a pharmaceutically acceptable salt thereof, or any pharmaceutical composition described herein. In some embodiments, the administration is by oral, parenteral, topical (dermal), or rectal administration. In some embodiments, any D-peptide described herein, or a pharmaceutically acceptable salt thereof, or any multimer described herein, or a pharmaceutically acceptable salt thereof, or any pharmaceutical composition described herein is administered topically to reduce TNFα activity or inflammation or inflammatory response. In some embodiments, the subject has a TNFα-mediated disease. In some embodiments, the TNFα-mediated disease is adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, cutaneous lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (polyarticular), hidradenitis suppurativa, uveitis (intermediate, posterior, or pan-uveitis), or radiographic negative axial spondyloarthritis.

[0041] In some embodiments, there is provided a D-peptide described herein, a multimer of any D-peptide described herein, or a salt thereof, for use as a medicament.

[0042] In some embodiments, there is provided a peptide described herein, a multimer of any D-peptide described herein, or a pharmaceutically acceptable salt thereof, for use in a method of treatment of a subject by therapy. In some embodiments, the subject has a TNFα-mediated disease. In some embodiments, the TNFα-mediated disease is adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, cutaneous lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (polyarticular), hidradenitis suppurativa, uveitis (intermediate, posterior, or pan-uveitis), or radiographic negative axial spondyloarthritis.

[0043] These and other aspects of the present application can be more fully understood in view of the following detailed description, the non-limiting examples of specific embodiments, and the appended drawings. BRIEF DESCRIPTION OF DRAWINGS

[0044] The foregoing aspects and many of the attendant advantages of this application will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0045] Figure 1 A comparison of the binding of selected peptides isolated from all libraries to TNFα by mirror phage screening is shown.

[0046] Figure 2 A comparison of the binding of peptide 18 and two peptide 18 variants to TNFα in a solid phase binding assay is shown.

[0047] Figure 3 It is shown that peptide 18 is able to block the binding of TNFα to its receptor TNFR1 in a solid phase binding assay.

[0048] Figure 4A It is shown that peptide 18 is able to block the cellular activity of TNFα in an L-929 cell killing assay.

[0049] Figure 4B It is shown that peptide 18 is able to block the cellular activity of TNFα in an L-929 cell killing assay in a concentration dependent manner.

[0050] Figure 5 The effect of amino acid changes at the N-terminal, C-terminal and internal positions of peptide 18 on TNFα binding is shown.

[0051] Figure 6 The effect of amino acid changes at the N-terminal, C-terminal and internal positions of peptide 18 using the TNFα / TNFR blocking assay is shown.

[0052] Figure 7 The effect of amino acid changes at the N-terminal, C-terminal and internal positions of peptide 18 using the TNFα / TNFR blocking assay is shown.

[0053] Figure 8 The effect of amino acid changes at the N-terminal, C-terminal and internal positions of peptide 18 using the L-929 cell killing assay is shown.

[0054] Figure 9 The effect of insertion of N-terminal PEG4 spacer and insertion or deletion of amino acid residues on peptide 18 using the TNFα binding assay is shown.

[0055] Figure 10 The effect of insertion of N-terminal PEG spacer and insertion or deletion of amino acid residues on peptide 18 using the L-929 cell killing assay is shown.

[0056] Figure 11Peptide 18 KtoR dimers and trimers were compared for activity using the L-929 cell killing assay.

[0057] Figure 12 Peptide 18 mutants and other library matches were compared for activity using the TNFα binding assay.

[0058] Figure 13 Peptide 18 KtoR trimers with different PEG lengths were compared for activity using the L-929 cell killing assay.

[0059] Figure 14 Peptide 18 and peptide 18 mutants were compared for activity using the L-929 cell killing assay.

[0060] Figure 15A The structure of three multimeric backbones used to make peptide trimers is shown.

[0061] Figure 15B Three peptide 18 KtoR trimers were compared for activity using the L-929 cell killing assay.

[0062] Figure 16A The structure of six multimeric backbones used to make peptide trimers is shown.

[0063] Figure 16B Six peptide 18 KtoR trimers were compared for activity using the L-929 cell killing assay.

[0064] Figure 17 Peptide 18 KtoR trimers with different PEG lengths were compared for activity using the L-929 cell killing assay.

[0065] Figure 18 Peptide 18 mutants were compared for activity using the L-929 cell killing assay.

[0066] Figure 19 The effect of replacing the N-terminal aspartic acid residue with succinic acid using the L-929 cell killing assay is shown.

[0067] Figure 20 The activity of peptide 18 mutants containing non-standard amino acids using the L-929 cell killing assay is shown.

[0068] Figure 21 Fluorescently labeled peptide DD-018 is shown binding to native TNFα.

[0069] Figure 22A and Figure 22B Peptide TF-018-KtoR-WW and its oligomers and anti-TNFα antibodies were compared for activity using the TNFα / TNFR blocking assay.

[0070] Figure 23A and Figure 23B Peptide TF-018-KtoR-WW and oligomer were compared to the activity of anti-TNFa antibody using L-929 cell killing assay.

[0071] Figure 24 Peptide TF-18-KtoR-WW C-trimer was shown to be able to block the activity of membrane bound TNFa.

[0072] Figure 25 Peptide TF-18-KtoR-WW C-trimer was shown to inhibit IL-8 levels after anti-CD28 antibody stimulation of blood samples. + Peptide TF-18-KtoR-WW C-trimer was shown to inhibit IL-8 levels after anti-CD28 antibody stimulation of blood samples.

[0073] Figure 26 Peptide TF-18-KtoR-WW C-trimer was shown to be able to block the activity of membrane bound TNFa.

[0074] Figure 27 and Figure 28 Peptide TF-18-KtoR-WW C-trimer was shown to be able to block the activity of membrane bound TNFa.

[0075] Figure 29 Peptide TF-18-KtoR-WW C-trimer was shown to be able to block the activity of membrane bound TNFa.

[0076] Figure 30 Peptide TF-18-KtoR-WW C-trimer was shown to be able to block the activity of membrane bound TNFa.

[0077] Figure 31 Peptide TF-18-KtoR-WW C-trimer was shown to be able to block the activity of membrane bound TNFa.

[0078] Figure 32 Peptide TF-18-KtoR-WW C-trimer was shown to be able to block the activity of membrane bound TNFa. Definitions

[0079] For convenience, certain terms used in the specification, examples, and appended claims are defined here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application, as the scope of the present application is limited only by the claims.

[0080] The terms "a" or "an", as used herein, are defined as "one" or "at least one." The singular form "a" or "an" includes plural references unless the context clearly dictates otherwise. The use of the term "at least one" followed by a list of one or more items should be interpreted as including only one item from the list.

[0081] Unless otherwise defined, all terms used in connection with the present disclosure, including technical and scientific terms, should be interpreted as is customary in the art or as given by recognized dictionaries (for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, New York; and Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York). The terms of degree such as "comprising", "including", "containing", etc. should be construed to be inclusive and not exclusive, unless expressly specified otherwise by context or by expressly set forth alternatives. Accordingly, these terms, where they appear in a specification written in English, should be construed to cover any of the applicable jurisdictions' equivalent facilitating provisions.

[0082] The terms "decrease", "reduce", "reduced", "reduction", and "inhibit" are used herein generally to mean a decrease by a statistically significant amount relative to a reference value.

[0083] The terms "increase", "enhance", or "activate" are used herein generally to mean an increase by a statistically significant amount relative to a reference value.

[0084] The terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues connected to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" also refer to amino acid polymers in which one or more amino acids are anabolic acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" generally refer to relatively large polypeptides, while the term "peptide" generally refers to small polypeptides, although there is some overlap in their use in the art. The terms "protein" and "polypeptide" are used interchangeably herein when referring to encoded gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0085] TNFα (also known as TNFalpha or TNF-alpha or TNF) is a protein expressed in a variety of cell types, including monocytes, macrophages, NK cells, and regulatory T cells. TNFα has both membrane and soluble forms. TNFα proteins include, but are not limited to, proteins having the amino acid sequences set forth in accession numbers AQY77150.1, P01375-1, and NP_000585.2; these sequences are incorporated herein by reference.

[0086] The terms "D-amino acid" and "D-amino acid residue" as used herein refer to an alpha-amino acid residue having the same absolute configuration as D-glyceraldehyde.

[0087] Amino acids as used herein are named herein according to the IUPAC recommendations, using either the one-letter or three-letter code. Unless otherwise specified by context, the amino acids are of the D form.

[0088] The phrases "D-form of a typical L-amino acid" and "D-form of any typical L-amino acid" refer to D-forms of alanine, arginine, asparagine, aspartic acid (aspartate), cysteine, glutamine, glutamic acid (glutamate), histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. For convenience, the phrase also refers to glycine, although this amino acid is non-chiral.

[0089] The term "D-peptide" as used herein refers to a peptide composed of D-amino acid residues.

[0090] "Specifically binds" as used herein refers to the ability of a binding agent (e.g., a D-peptide described herein) to bind to a target with a KDof 10 -5 M (10000 nM) or lower (e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or lower). Specific binding can be influenced by, for example, the affinity and avidity of the binding agent and the concentration of the target polypeptide. One of ordinary skill in the art can determine appropriate conditions under which a binding agent selectively binds to a target using any suitable method (e.g., titrating the binding agent in a suitable cell binding assay or a suitable solid phase binding assay). A binding agent that specifically binds to a target is not displaced by a non-analogous competitor. In certain embodiments, a binding agent is said to specifically bind to its target when it preferentially recognizes its target in a complex mixture of proteins and / or macromolecules.

[0091] The term "consisting essentially of as used herein refers to those elements required for the particular embodiments. The term permits the inclusion of elements that do not materially affect the basic and novel or functional characteristic of the embodiment.

[0092] The term "comprising" as used herein is intended to mean that the compositions, methods, and respective components thereof described herein can include additional elements not listed or consisting only of components described. The term "consisting essentially of as used herein refers to those elements required for the particular embodiments. The term permits the inclusion of elements that do not materially affect the basic and novel or functional characteristic of the embodiment.

[0093] All numbers expressing quantities of ingredients, reaction conditions, and so forth used herein are to be understood as being modified in all instances by the term "about," unless otherwise indicated. The term "about" when used in connection with a percentage, can mean + / - 1%.

[0094] The term "statistically significant" or "significant" refers to statistical significance, generally meaning a difference of two standard deviations (2SD), above or below a reference value.

[0095] The term "pharmaceutically acceptable salt" refers generally to salts that retain the biological effectiveness and properties of the free bases and that are not biologically or otherwise undesirable. "Pharmaceutically acceptable salts" include solvates, especially hydrates, of such salts.

[0096] Other terms are defined in the description of the various aspects of the application. DETAILED DESCRIPTION

[0097] The present disclosure is based on the discovery of a class of potent and specific D-peptide-based TNFa inhibitors (also referred to herein as TNFi peptides). In some embodiments, the peptides inhibit TNFa activity by binding to TNFa. In some embodiments, the peptides inhibit TNFa activity by binding to TNFa and blocking its binding to its receptors, TNFR1 and / or TNFR2. In some embodiments, the peptides inhibit TNFa activity by blocking its binding to TNFR1. In some embodiments, the peptides inhibit TNFa activity by blocking its binding to TNFR2. The TNFi peptides comprise a core TNFa binding domain composed of D-amino acids. In some embodiments, the TNFi peptides specifically bind to both soluble TNFa and membrane-bound TNFa. In some embodiments, the TNFi peptides specifically bind to soluble TNFa. In some embodiments, the TNFi peptides specifically bind to membrane-bound TNFa. In some embodiments, the TNFi-peptides are resistant to proteolysis in the gastrointestinal environment.

[0098] Also provided are methods of using TNFi peptides to treat TNFα-mediated diseases, including inflammatory and autoimmune diseases and conditions. As used herein, the term "TNFα-mediated disease" refers to a disease or condition in which the disease or its symptoms involve the TNFα signaling pathway and / or cellular biological effects of TNFα. In some embodiments, the TNFi peptides are administered by parenteral administration, intravenous administration, intramuscular administration, subcutaneous administration, oral administration, topical administration, or rectal administration. These and other embodiments will be described further herein.

[0099] Peptides

[0100] Provided herein are D-peptide-based inhibitors of TNFα. In some embodiments, the peptides inhibit TNFα activity by binding to TNFα. In some embodiments, the peptides inhibit TNFα activity by binding to TNFα and blocking its binding to its receptors, TNFR1 and / or TNFR2. The TNFi peptides comprise a core TNFα binding domain composed of D-amino acids.

[0101] In some embodiments, provided are D-peptides or salts thereof, wherein the peptide comprises a core TNFα binding domain of D-amino acids and has the amino acid sequence, C-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -C (SEQ ID NO: 111), wherein each of X 2 to X 11 is a D-amino acid; and X 2 is the D form of any typical L-amino acid except cysteine; X 3 is the D form of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), or Leu (L); X 4 is a polar amino acid including Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gin (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G); X 5 is the D form of any typical L-amino acid except cysteine; X 6 is the D form of any typical L-amino acid except cysteine; X 7 is the D form of Phe (F); X 8 is the D form of Asn (N), X9 is D-Asn (N), X 10 is D-Asn (N), X 11 is D-Asn (N), X

[0102] In some embodiments, a D-peptide or salt thereof is provided, wherein the peptide comprises a core TNFα binding domain of D-amino acids and has the amino acid sequence, C*-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -C*(SEQ ID NO: 112), wherein X 2 to X 11 each is a D-amino acid; and X 2 is the D-form of any typical L-amino acid except cysteine; X 3 is the D-form of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), and Leu (L); X 4 is a polar amino acid including Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gln (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), and Gly (G); X 5 is the D-form of any typical L-amino acid except cysteine; X 6 is the D-form of any typical L-amino acid except cysteine; X 7 is the D-form of Phe (F); X 8 is D-Asn (N), X 9 is D-Asn (N), X 10 is D-Asn (N), X 11 is the D-form of Trp (W), Tyr (Y), Gln (Q), or His (H); C represents the D-form of cysteine; and * represents an optional intramolecular bond.

[0103] In some embodiments, a D-peptide or salt thereof is provided, wherein the peptide comprises a core TNFα binding domain of D-amino acids and has the amino acid sequence, C*-X 2 -[W / F / Y]-[Polar]-X 5 -X 6-F-N-N-[W / Y]-W-C*(SEQ ID NO: 113), wherein X 2 is the D form of any typical L-amino acid other than Cys, X 5 is the D form of any typical L-amino acid other than Cys, X 6 is the D form of any typical L-amino acid other than Cys, Polar represents a D-amino acid comprising one of R, K, H, E, D, Q, N, T, S, P, A, or G, and * represents an optional intramolecular disulfide bond.

[0104] In some embodiments, the TNFi peptide comprises a core TNFα binding domain of D-amino acids and has the following amino acid sequence: C-X 2 -X 3 -X 4 -X 5 -X 6 -F-N-N-X 10 -X 11 -C(SEQ ID NO: 1), wherein X 1 to X 6 , X 10 and X 11 each are D-amino acids; and X 2 is D-Thr (T), Val (V), His (H), Leu (L), Gin (Q), Ala (A), He (I), Met (M), or Trp (W), or a D-a-amino acid analog thereof; X 3 is D-Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), or Leu (L), or a D-a-amino acid analog thereof; X 4 is D-Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gin (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G), or a D-a-amino acid analog thereof; X 5 is D-Pro (P), Trp (W), His (H), Gin (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), or a D-a-amino acid analog thereof; X 6 is D-Arg (R), His (H), Lys (K), Glu (E), Gin (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-a-amino acid analog thereof; X 10 is D-Trp (W) or Tyr (Y), or a D-a-amino acid analog thereof; X 11is D-Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), lie (I), Met (M), or Trp (W), or a D-a-amino acid analog thereof; and X

[0105] In some embodiments, the TNFi peptide comprises a core TNF a binding domain of D-amino acids and has the following amino acid sequence: C*-X 2 -X 3 -X 4 -X 5 -X 6 -F-N-N-X 10 -X 11 -C*(SEQ ID NO: 2), wherein X 1 each of X 6 , X 10 , and X 11 is a D-amino acid; and X 2 is D-Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), lie (I), Met (M), or Trp (W), or a D-a-amino acid analog thereof; and X 3 is D-Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), or Leu (L), or a D-a-amino acid analog thereof; X 4 is a polar amino acid comprising D-Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gln (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G), or a D-a-amino acid analog thereof; X 5 is D-Pro (P), Trp (W), His (H), Gln (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), or a D-a-amino acid analog thereof; X 6 is D-Arg (R), His (H), Lys (K), Glu (E), Gln (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-a-amino acid analog thereof; X 10 is D-Trp (W) or Tyr (Y), or a D-a-amino acid analog thereof; X 11 is D-Trp (W), Tyr (Y), Gln (Q), or His (H); C represents D-cysteine; F represents D-phenylalanine; N represents D-asparagine; and * represents an optional intramolecular disulfide bond.

[0106] In some embodiments of the TNFi peptide set forth in SEQ ID NO: 1 or 2, X2 is D-form Thr, Val, His, Leu, Gin, Ala, lie, Met, or Trp. In some embodiments, X 2 is D-form Thr, Val, His, Leu, or Gin. In some embodiments, X 2 is D-form Thr, Val, His, or Leu. In some embodiments, X 2 is D-form Thr, Val, or His. In some embodiments, X 2 is D-form Thr or Val. In some embodiments, X 2 is D-form Thr. In some embodiments, X 2 is D-form Val.

[0107] In some embodiments of the TNFi peptide set forth in SEQ ID NO: 1 or 2, X 3 is D-form Trp, Phe, Tyr, or Ser. In some embodiments, X 3 is D-form Trp, Phe, or Tyr. In some embodiments, X 3 is D-form Trp or Phe. In some embodiments, X 3 is D-form Trp. In some embodiments, X 3 is D-form Phe.

[0108] In some embodiments of the TNFi peptide set forth in SEQ ID NO: 1 or 2, X 4 is D-form Arg, His, Gin, Lys, Asn, Thr, or Ser. In some embodiments, X 4 is D-form Arg, His, Gin, Lys, or Asn. In some embodiments, X 4 is D-form Arg, His, or Gin. In some embodiments, X 4 is D-form Arg, Gin, or Asn. In some embodiments, X 4 is D-form Gin.

[0109] In some embodiments of the TNFi peptide set forth in SEQ ID NO: 1 or 2, X 5 is D-form Pro, Trp, His, Gin, Gly, Arg, Ala, Val, or Leu. In some embodiments, X 5 is D-form Pro, Trp, His, Gin, Gly, Arg, or Val. In some embodiments, X 5 is D-form Pro, Trp, or His. In some embodiments, X 5 is D-form Pro or Trp. In some embodiments, X5 is D-form Pro.

[0110] In some embodiments of the TNFi peptide set forth in SEQ ID NO: 1 or 2, X 6 is D-form Arg, His, Lys, Glu, Gin, Val, Leu, Ser, or Ala. In some embodiments, X 6 is D-form Arg, His, Lys, Glu, Gin, Val, or Leu. In some embodiments, X 6 is D-form Arg, His, Lys, Glu, or Gin. In some embodiments, X 6 is D-form Arg, His, Lys, or Glu. In some embodiments, X 6 is D-form Arg, Lys, or His. In some embodiments, X 6 is D-form Arg. In some embodiments, X 6 is D-form Lys. In some embodiments, X 6 is D-form His.

[0111] In some embodiments of the TNFi peptide set forth in SEQ ID NO: 1 or 2, X 10 is D-form Trp. In some embodiments, X 10 is D-form Tyr.

[0112] In some embodiments of the TNFi peptide set forth in SEQ ID NO: 1 or 2, X 11 is D-form Trp (W), Tyr (Y), or Gin (Q). In some embodiments, X 11 is D-form Trp (W).

[0113] In some embodiments, the TNFi peptide comprises a core TNFα binding domain of D-amino acids and has the following amino acid sequence: C*-X 2 -[W / F / Y]-X 4 -X 5 -X 6 -F-N-N-[W / Y]-W-C* (SEQ ID NO: 3), wherein X 2 is D-form Thr (T), Val (V), His (H), Leu (L), Gin (Q), Ala (A), lie (I), Met (M), or Trp (W); X 4 is D-form Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gin (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G); X 5is D-form Pro (P), Trp (W), His (H), Gin (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), X 6 is D-form Arg (R), His (H), Lys (K), Glu (E), Gin (Q), Val (V), Leu (L), Ser (S), or Ala (A); and * indicates an optional intramolecular disulfide bond between the indicated cysteine residues.

[0114] In some embodiments of the TNFi peptide set forth in SEQ ID NO: 3, X 2 is D-form Thr, Val, His, Leu, Gin, Ala, He, Met, or Trp. In some embodiments, X 2 is D-form Thr, Val, His, Leu, or Gin. In some embodiments, X 2 is D-form Thr, Val, His, or Leu. In some embodiments, X 2 is D-form Thr, Val, or His. In some embodiments, X 2 is D-form Thr or Val. In some embodiments, X 2 is D-form Thr. In some embodiments, X 2 is D-form Val.

[0115] In some embodiments of the TNFi peptide set forth in SEQ ID NO: 3, X 4 is selected from D-form Arg, His, Lys, Gin, Asn, Thr, or Ser. In some embodiments, X 4 is D-form Arg, His, Lys, Gin, or Asn. In some embodiments, X 4 is D-form Arg, His, Lys, or Gin. In some embodiments, X 4 is D-form Arg, Gin, or Asn. In some embodiments, X 4 is D-form Arg, Gin, or Lys. In some embodiments, X 4 is D-form Gin.

[0116] In some embodiments of the TNFi peptide set forth in SEQ ID NO: 3, X 5 is D-form Pro, Trp, His, Gin, Gly, Arg, Ala, Val, or Leu. In some embodiments, X 5 is D-form Pro, Trp, His, Lys, Gin, Gly, Arg, or Val. In some embodiments, X 5is D-form Pro, Trp, or His. In some embodiments, X 5 is D-form Pro or Trp. In some embodiments, X 5 is D-form Pro.

[0117] In some embodiments of the TNFi peptide set forth in SEQ ID NO: 3, X 6 is D-form Arg, His, Lys, Glu, Gin, Val, Leu, Ser, or Ala. In some embodiments, X 6 is D-form Arg, His, Lys, Glu, Gin, Val, or Leu. In some embodiments, X 6 is D-form Arg, His, Lys, Glu, or Gin. In some embodiments, X 6 is selected from D-form Arg, His, Lys, or Glu. In some embodiments, X 6 is D-form Arg or His. In some embodiments, X 6 is D-form Arg or Lys. In some embodiments, X 6 is D-form Arg. In some embodiments, X 6 is D-form His. In some embodiments, X 6 is D-form Lys.

[0118] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the amino acid sequence: CVWQPKFNNYWC (SEQ ID NO: 4), optionally comprising an intramolecular disulfide bond.

[0119] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the amino acid sequence: CVWQPRFNNYWC (SEQ ID NO: 5), optionally comprising an intramolecular disulfide bond.

[0120] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the amino acid sequence: CTFQPRFNNYWC (SEQ ID NO: 6), optionally comprising an intramolecular disulfide bond.

[0121] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the amino acid sequence: CTFQPRFNNWWC (SEQ ID NO: 7), optionally comprising an intramolecular disulfide bond.

[0122] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the amino acid sequence: CSFQPRFNNYWC (SEQ ID NO: 8), optionally comprising an intramolecular disulfide bond.

[0123] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CSFQPRFNNWWC (SEQ ID NO: 9), optionally comprising an intramolecular disulfide bond.

[0124] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFQPRFNNYWC (SEQ ID NO: 10), optionally comprising an intramolecular disulfide bond.

[0125] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFQPRFNNWWC (SEQ ID NO: 11), optionally comprising an intramolecular disulfide bond.

[0126] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTFQWRFNNYWC (SEQ ID NO: 12), optionally comprising an intramolecular disulfide bond.

[0127] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CLYQPVFNNWWC (SEQ ID NO: 13), optionally comprising an intramolecular disulfide bond.

[0128] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFQAAFNNYWC (SEQ ID NO: 14), optionally comprising an intramolecular disulfide bond.

[0129] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFQHHFNNWWC (SEQ ID NO: 15), optionally comprising an intramolecular disulfide bond.

[0130] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CHFNPRFNNWWC (SEQ ID NO: 16), optionally comprising an intramolecular disulfide bond.

[0131] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVWQPHFNNYWC (SEQ ID NO: 17), optionally comprising an intramolecular disulfide bond.

[0132] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFQGRFNNWWC (SEQ ID NO: 18), optionally comprising an intramolecular disulfide bond.

[0133] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFQHRFNNWWC (SEQ ID NO: 19), optionally comprising an intramolecular disulfide bond.

[0134] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFNPRFNNWWC (SEQ ID NO: 20), optionally comprising an intramolecular disulfide bond.

[0135] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFKPRFNNWWC (SEQ ID NO: 21), optionally comprising an intramolecular disulfide bond.

[0136] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CAYQRQFNNWWC (SEQ ID NO: 22), optionally comprising an intramolecular disulfide bond.

[0137] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CWFEHRFNNWHC (SEQ ID NO: 23), optionally comprising an intramolecular disulfide bond.

[0138] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CHFQHRFNNWWC (SEQ ID NO: 24), optionally comprising an intramolecular disulfide bond.

[0139] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CHFQPRFNNWWC (SEQ ID NO: 25), optionally comprising an intramolecular disulfide bond.

[0140] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTYQPRFNNWWC (SEQ ID NO: 26), optionally comprising an intramolecular disulfide bond.

[0141] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CQFQPRFNNWQC (SEQ ID NO: 27), optionally comprising an intramolecular disulfide bond.

[0142] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CHFSQRFNNWWC (SEQ ID NO: 28), optionally comprising an intramolecular disulfide bond.

[0143] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CIFQQQFNNYWC (SEQ ID NO: 77), optionally comprising an intramolecular disulfide bond.

[0144] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CMHQQRFNNWWC (SEQ ID NO: 78), optionally comprising an intramolecular disulfide bond.

[0145] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTFRVRFNNYWC (SEQ ID NO: 79), optionally comprising an intramolecular disulfide bond.

[0146] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CIFQWRFNNYWC (SEQ ID NO: 80), optionally comprising an intramolecular disulfide bond.

[0147] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTFQWHFNNYWC (SEQ ID NO: 81), optionally comprising an intramolecular disulfide bond.

[0148] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFQHLFNNWWC (SEQ ID NO: 82), optionally comprising an intramolecular disulfide bond.

[0149] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTFQWLFNNYWC (SEQ ID NO: 83), optionally comprising an intramolecular disulfide bond.

[0150] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CAFQWRFNNYWC (SEQ ID NO: 84), optionally comprising an intramolecular disulfide bond.

[0151] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CAFQWRFNNYWC (SEQ ID NO: 84), optionally comprising an intramolecular disulfide bond.

[0152] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTSQWRFNNYWC (SEQ ID NO: 86), optionally comprising an intramolecular disulfide bond.

[0153] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTSQWRFNNYWC (SEQ ID NO: 86), optionally comprising an intramolecular disulfide bond.

[0154] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTSQWRFNNYWC (SEQ ID NO: 86), optionally comprising an intramolecular disulfide bond.

[0155] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTSQWRFNNYWC (SEQ ID NO: 86), optionally comprising an intramolecular disulfide bond.

[0156] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTSQWRFNNYWC (SEQ ID NO: 86), optionally comprising an intramolecular disulfide bond.

[0157] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTSQWRFNNYWC (SEQ ID NO: 86), optionally comprising an intramolecular disulfide bond.

[0158] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTSQWRFNNYWC (SEQ ID NO: 86), optionally comprising an intramolecular disulfide bond.

[0159] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVSTHHFNNWWC (SEQ ID NO: 93), optionally comprising an intramolecular disulfide bond.

[0160] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CAYQRHFNNWWC (SEQ ID NO: 95), optionally comprising an intramolecular disulfide bond.

[0161] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CAYQRHFNNWWC (SEQ ID NO: 95), optionally comprising an intramolecular disulfide bond.

[0162] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CHFNPLFNNWWC (SEQ ID NO: 96), optionally comprising an intramolecular disulfide bond.

[0163] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CHFNPLFNNWWC (SEQ ID NO: 96), optionally comprising an intramolecular disulfide bond.

[0164] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CHFSQLFNNWWC (SEQ ID NO: 98), optionally comprising an intramolecular disulfide bond.

[0165] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CHFSQLFNNWWC (SEQ ID NO: 98), optionally comprising an intramolecular disulfide bond.

[0166] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CQFRPRFNNWQC (SEQ ID NO: 100), optionally comprising an intramolecular disulfide bond.

[0167] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CQFRPRFNNWQC (SEQ ID NO: 100), optionally comprising an intramolecular disulfide bond.

[0168] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTSRWRFNNYWC (SEQ ID NO: 102), optionally comprising an intramolecular disulfide bond.

[0169] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFQASFNNYWC (SEQ ID NO: 103), optionally comprising an intramolecular disulfide bond.

[0170] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFQHSFNNWWC (SEQ ID NO: 104), optionally comprising an intramolecular disulfide bond.

[0171] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFRHHFNNWWC (SEQ ID NO: 105), optionally comprising an intramolecular disulfide bond.

[0172] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFTHHFNNWWC (SEQ ID NO: 106), optionally comprising an intramolecular disulfide bond.

[0173] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVSQHHFNNWWC (SEQ ID NO: 107), optionally comprising an intramolecular disulfide bond.

[0174] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVWQPEFNNYWC (SEQ ID NO: 108), optionally comprising an intramolecular disulfide bond.

[0175] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVWQQKFNNYWC (SEQ ID NO: 109), optionally comprising an intramolecular disulfide bond.

[0176] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVWRPKFNNYWC (SEQ ID NO: 110), optionally comprising an intramolecular disulfide bond.

[0177] In some embodiments, the TNFi peptide comprises a core TNFα binding domain selected from the group consisting of: CVWQPKFNNYWC (SEQ ID NO:4); CVWQPRFNNYWC (SEQ ID NO:5); CTFQPRFNNYWC (SEQ ID NO:6); CTFQPRFNNWWC (SEQ ID NO:7); CSFQPRFNNYWC (SEQ ID NO:8); CSFQPRFNNWWC (SEQ ID NO:9); CVFQPRFNNYWC (SEQ ID NO: 10); CVFQPRFNNWWC (SEQ ID NO: 11); CTFQWRFNNYWC (SEQ ID NO: 12); CHFNPRFNNWWC (SEQ ID NO: 16); CVFQGRFNNWWC (SEQ ID NO: 18); CVFQHRFNNWWC (SEQ ID NO: 19); CVFNPRFNNWWC (SEQ ID NO:20); CVFKPRFNNWWC (SEQ ID NO:21); optionally comprising an intramolecular disulfide bond in each case.

[0178] In some embodiments, the TNFi peptide comprises a core TNFα binding domain selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs:4-28 and 77-110.

[0179] TNFi peptides containing D-amino acid analogs

[0180] In some embodiments, one or more D-amino acids in a TNFi peptide can be replaced with a D-a-amino acid analog of a D-amino acid. A D-a-amino acid analog is a D-a-amino acid analog that possesses both an amine functional group (NH2, NHR, or NR2) and a carboxylic acid functional group. In some embodiments, a TNFi peptide having a core TNFα binding domain set forth in any one of SEQ ID NOs: 1 to 3 and 111 to 113 has at least one D-a-amino acid analog substitution in the core TNFα binding domain. In some embodiments, a TNFi peptide having a core TNFα binding domain set forth in any one of SEQ ID NOs: 1 to 3 and 111 to 113 has at least two D-a-amino acid analog substitutions in the core TNFα binding domain. In some embodiments, a TNFi peptide having a core TNFα binding domain set forth in any one of SEQ ID NOs: 1 to 3 and 111 to 113 has at least three D-a-amino acid analog substitutions in the core TNFα binding domain.

[0181] Charged D-a-amino acid analogs include D-type 4-methyl glutamic acid, mono-4- fluoroglutamic acid, 4,4-difluoroglutamic acid, gamma-hydroxy glutamic acid, L-threo-beta- hydroxy aspartic acid, epsilon-N,N,N-trimethyl lysine, epsilon-N-acetyl lysine, azallysine, O- phosphoserine, 3-methylhistidine, 5-hydroxy lysine, and methylarginine.

[0182] Polar uncharged D-amino acid analogs include D-type L-Glu gamma-hydrazide, L- acacetic acid, L-theophylline, beta-hydroxy norvaline, aspartic acid methyl ester, and glutamic acid methyl ester.

[0183] Aromatic D-amino acid analogs include D-type beta-2-thiazolylalanine, triazolylalanine, 3-fluoro-L-tyrosine, 3-nitro-L-tyrosine, 3-fluorophenylalanine, 2-thienylalanine, beta- methylphenylalanine, beta-thienylserine, N-acetylserine, N-formylmethionine, p-azidophenylalanine, p-ethynylphenylalanine, p-nitrophenylalanine, 7-azatryptophan, 5-hydroxytryptophan, 5- fluorotryptophan, 5-methoxytryptophan, 3-(thianapthen-3-yl)-L-alanine, 2-thienylglycine, and L-phenylglycine.

[0184] Nonpolar D-amino acid analogs include D-type 2-amino hex-5-ynoic acid, norleucine, norvaline, ethionine, beta-azidohomoalanine, trifluoro-norleucine, trifluoro-norvaline, L-C- propargylglycine, L-allylglycine, beta-cyclopropylalanine, 3-fluorovaline, methyloxymethyl-L- threonine, methyloxymethyl-L-allothreonine, 4-thia-isoleucine, L-cyclohexyl-glycine, 5',5',5'- trifluoroleucine, beta-cyclopentylalanine, thiazolidine-2-carboxylic acid, thiazolidine-4- carboxylic acid, pseutdoproline, 3,4-dehydroproline, and 4-hydroxyproline.

[0185] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having D-a-amino acid analogs having the following amino acid sequence: XTFQPRFNNWWC (SEQ ID NO: 114), optionally comprising an intramolecular disulfide bond, wherein X is penicillamine (Pen).

[0186] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having D-a-amino acid analogs having the following amino acid sequence: CTFQPRFNNWWX (SEQ ID NO: 115), optionally comprising an intramolecular disulfide bond, wherein X is penicillamine (Pen).

[0187] In some embodiments, the TNFi peptide comprises a core TNFα binding domain with D-α-amino acid analogs having the following amino acid sequence: CXFQPRFNNWWC (SEQ ID NO: 74), optionally comprising an intramolecular disulfide bond, wherein X is homoLeu.

[0188] In some embodiments, the TNFi peptide comprises a core TNFα binding domain with D-α-amino acid analogs having the following amino acid sequence: CXFQPRFNNWWC (SEQ ID NO: 75), optionally comprising an intramolecular disulfide bond, wherein X is norLeu.

[0189] Other modifications of TNFi peptides

[0190] In some embodiments, the TNFi peptide is capped at the N-terminus and / or C-terminus. In some embodiments, the TNFi peptide is capped at the N-terminus. In some embodiments, the TNFi peptide is capped at the N-terminus with, e.g., an acetyl group. In some embodiments, the TNFi peptide is capped at the N-terminus with, e.g., a protecting group. In some embodiments, the TNFi peptide is capped at the C-terminus. In some embodiments, the TNFi peptide is capped at the C-terminus with, e.g., an amide group. In some embodiments, the TNFi peptide is capped at the C-terminus with, e.g., a protecting group.

[0191] In some embodiments, the TNFi peptide has a core TNFα binding domain flanked by additional amino acid residues (flanking sequences or tags) attached to the N-terminus and / or C-terminus. In some embodiments, the TNFi peptide has a core TNFα binding domain flanked by additional D-amino acid residues (flanking sequences or tags) attached to the N-terminus and / or C-terminus. The tags, for example, can increase the solubility of the TNFi peptide (e.g., in aqueous solution). When there is more than one tag, the tags can be the same or different. In some embodiments, a tag is attached to the N-terminus of the core TNFα binding domain. In some embodiments, a tag is attached to the C-terminus of the core TNFα binding domain. In some embodiments, a tag is attached to the N-terminus and to the C-terminus of the core TNFα binding domain.

[0192] In some embodiments, a tag is attached to the N-terminus of the core TNFa binding domain. In some embodiments, a tag attached to the N-terminus of the core TNFa binding domain is 1 to 20 amino acids in length, 1 to 10 amino acids in length, 1 to 6 amino acids in length, or 1 to 3 amino acids in length, 2 to 3 amino acids in length, or 1 to 2 amino acids in length, or 2 amino acids in length. In some embodiments, a tag of 1, 2, or 3 D-aspartate residues (D, DD, or DDD, respectively) is attached to the N-terminus of the core-TNFa peptide binding domain. In some embodiments, a tag of the amino acid sequence DGA is attached to the N-terminus of the core TNFa binding domain. In some embodiments, a tag of 1 or 2 D-aspartate residues is attached to the N-terminus of the core TNFa binding domain. In some embodiments, a tag of 2 D-aspartate residues is attached to the N-terminus of the core TNFa binding domain. In some embodiments, a tag of 1 D-aspartate residue is attached to the N-terminus of the core TNFa binding domain.

[0193] In some embodiments, a tag is attached to the C-terminus of the core TNFa binding domain. In some embodiments, a tag attached to the C-terminus of the core TNFa binding domain is 1 to 10 amino acids in length, or 1 to 8 amino acids in length, 2 to 8 amino acids in length, or 2 to 6 amino acids in length. In some embodiments, a tag comprises a single glycine residue attached to the C-terminus of the core TNFa binding domain. In some embodiments, a tag comprises a pair of glycine residues attached to the C-terminus of the core TNFa binding domain. In some embodiments, a tag has the D-amino acid sequence GGEEEK (SEQ ID NO: 30) and is attached to the C-terminus of the core TNFa binding domain. In some embodiments, a tag has the D-amino acid sequence GGRRRK (SEQ ID NO: 31) and is attached to the C-terminus of the core TNFa binding domain.

[0194] In some embodiments, a TNFi peptide is capped at the N-terminus and / or C-terminus of a tag. In some embodiments, a TNFi peptide is capped at the N-terminus of a tag. In some embodiments, a TNFi peptide is capped at the N-terminus with, for example, an acetyl group. In some embodiments, a TNFi peptide is capped at the C-terminus of a tag. In some embodiments, a TNFi peptide is capped at the C-terminus with, for example, an amide group.

[0195] In some embodiments, the TNFi peptide can comprise additional amino acids in addition to the core TNFα binding domain and any attachment tag. These additional amino acids can be D-amino acids or L-amino acids. In some embodiments, the TNFi peptide, including the N-terminal and / or C-terminal tag, is 12 to 50 amino acids in length, or 12 to 40 amino acids in length, or 12 to 30 amino acids in length, or 12 to 20 amino acids in length. In some embodiments, the TNFi peptide, including the N-terminal and / or C-terminal tag, is 12 to 50 amino acids in length. In some embodiments, the TNFi peptide, including the N-terminal and / or C-terminal tag, is 12 to 40 amino acids in length. In some embodiments, the TNFi peptide, including the N-terminal and / or C-terminal tag, is 12 to 30 amino acids in length. In some embodiments, the TNFi peptide, including the N-terminal and / or C-terminal tag, is 12 to 20 amino acids in length.

[0196] Linkers

[0197] In some embodiments, the TNFi peptide comprises a linker attached to its N-terminus and / or its C-terminus. In some embodiments, the linker comprises L-amino acids. In some embodiments, the linker comprises D-amino acids. In some embodiments, the linker comprises a chemical group other than an amino acid. In some embodiments, the linker comprises both a chemical group (other than an amino acid) and an amino acid. In some embodiments, the linker comprises both a chemical group (other than an amino acid) and L-amino acids. In some embodiments, the linker comprises both a chemical group (other than an amino acid) and D-amino acids.

[0198] In some embodiments, the linker comprises a repeating polymer unit. In some embodiments, the polymer unit is attached to the N-terminus of the TNFi peptide. In some embodiments, the polymer unit is attached to the C-terminus of the TNFi peptide. In some embodiments, the polymer unit is attached to the N-terminus and to the C-terminus of the TNFi peptide. The polymer units attached to the N-terminus and the C-terminus can be the same or different.

[0199] In some embodiments, the polymer unit comprises a polyethylene glycol chain (PEG group). In some embodiments, the polymer unit comprises a polysaccharide chain. In some embodiments, the polymer unit comprises an alkyl polyol chain. In some embodiments, the polymer unit comprises an elastin-like polypeptide. In some embodiments, the polymer unit comprises a poly sarcosine chain. In some embodiments, the polymer unit comprises an alkyl chain. In some embodiments, the polymer unit comprises a polypeptide chain, such as albumin or other polypeptides.

[0200] In some embodiments, the linker comprises a polymeric unit having a polyethylene glycol chain (PEG group). In some embodiments, the linker comprises a linear or branched polyethylene glycol chain (PEG group). In some embodiments, the PEG group is attached to the N-terminus of the TNFi peptide. In some embodiments, the PEG group is attached to the C-terminus of the TNFi peptide. In some embodiments, the PEG group is attached to the N-terminus and to the C-terminus of the TNFi peptide. The PEG groups attached to the N-terminus and C-terminus can be the same or different.

[0201] In some embodiments, the PEG group has 1 to 48 (ethylene glycol) subunits, and is linear or branched. In some embodiments, the PEG group has 1 to 30 subunits, and is linear or branched. In some embodiments, the PEG group has 1 to 24 subunits, and is linear or branched. In some embodiments, the PEG group has 1 to 12 subunits. In some embodiments, the PEG group has 4 to 30 subunits, and is linear or branched. In some embodiments, the PEG group has 4 to 24 subunits, and is linear or branched. In some embodiments, the PEG group has 4 to 12 subunits, and is linear or branched. In some embodiments, the PEG group has 4 to 10 subunits, and is linear or branched. In some embodiments, the PEG group has 4 subunits, and is linear or branched. In some embodiments, the PEG group has 6 subunits, and is linear or branched. In some embodiments, the PEG group has 8 subunits, and is linear or branched. In some embodiments, the PEG group has 10 subunits, and is linear or branched. In some embodiments, the PEG group has 12 subunits, and is linear or branched.

[0202] In some embodiments, the linker comprises a functional group at the end of the linker opposite the site of attachment to the TNFi peptide. The functional group can serve as a cap and / or provide a site of attachment for another molecule, such as a multimeric scaffold group or a helper molecule. In some embodiments, the functional group is, for example, an acetate group, a carboxylic acid group, an amine group, an amide group, a carboxamide group, a thiol group, a hydrazide group, an NHS ester, or a pentafluorophenyl ester. In some embodiments, the functional group is one or more amino acid residues. In some embodiments, the one or more amino acid residues of the functional group are in the L-configuration. In some embodiments, the amino acid residues of the functional group are in the D-configuration. In some embodiments, the functional group is an amino acid residue, such as a lysine residue. In some embodiments, the functional group is a site of attachment for a multimeric scaffold. In some embodiments, the functional group comprises one or more lysine residues, one or more hydrazide groups, or one or more thiol or other groups.

[0203] Multimers

[0204] In some embodiments, the TNFi peptides can be in the form of a multimer, e.g., a dimer or a trimer or a more multimer. For example, when the multimer is a dimer, the dimer can be composed of two identical TNFi peptides (i.e., a homodimer) or two different TNFi peptides (i.e., a heterodimer). The multimer can also be a trimer. When the multimer is a trimer, the trimer can be composed of two identical TNFi peptides and one different TNFi peptide (i.e., a heterotrimer) or three identical TNFi peptides (i.e., a homotrimer) or three different TNFi peptides, each different from the others (i.e., a heterotrimer).

[0205] In some embodiments, two or more TNFi peptides can be linked by a linker (e.g., a linker of amino acid residues or other chemical moieties, as described herein or known to one of skill in the art) to form a multimer, e.g., a dimer, a trimer or a more multimer.

[0206] In some embodiments, the TNFi peptides form a dimer. In some embodiments, the TNFi peptides form a trimer. In some embodiments, the TNFi peptides form a multimer comprising at least four TNFi peptides. In some embodiments, a more multimer can comprise 5, 6, 7, 8, 9, 10, 11, 12 or more TNFi peptides. The multimer can be heteromeric or homomeric.

[0207] In some embodiments, the TNFi peptides are linked in a dimeric form and form a linear chain. In some embodiments, the TNFi peptides are linked together by the C-terminus of one peptide to the N-terminus of a second peptide, optionally comprising a linker connecting the two TNFi peptides. In some embodiments, the TNFi peptides are linked together by the C-terminus of one peptide to the C-terminus of a second peptide, optionally comprising a linker connecting the two TNFi peptides. In some embodiments, the TNFi peptides are linked together by the N-terminus of one peptide to the N-terminus of a second peptide, optionally comprising a linker connecting the two TNFi peptides. In any of these embodiments, the TNFi peptides can be the same or different.

[0208] In some embodiments, the TNFi peptides are linked in a trimeric form and form a linear chain. In some embodiments, the TNFi peptides are linked together by the C-terminus of one peptide to the N-terminus of a next peptide, the C-terminus of the next peptide to the N-terminus of a third peptide, optionally comprising a linker connecting pairs of TNFi peptides. In some embodiments, the TNFi peptides are linked in any suitable N-terminal and C-terminal orientation. In any of these embodiments, the TNFi peptides can be the same or different.

[0209] In some embodiments, TNFi peptides are linked in linear multimers, comprise at least four TNFi peptides, and form a linear chain. In some embodiments, more complex multimers can comprise 5, 6, 7, 8, 9, 10, 11, 12, or more peptides per chain. In some embodiments, multimers are linked together through the C-terminus of one peptide to the N-terminus of the next peptide, optionally comprising a linker connecting pairs of TNFi peptides. In some embodiments, multimers are linked in any suitable N- and C-terminal orientation, optionally comprising a linker connecting pairs of TNFi peptides. In any of these embodiments, the peptides can be the same or different.

[0210] In some embodiments, TNFi peptides are cross-linked to form multimers, such as branched multimers. TNFi peptides are cross-linked through their N-terminus and / or C-terminus. In some embodiments, the cross-linking agent is a polyethylene glycol (PEG) group derivatized with reactive groups, such as N-hydroxysuccinimide (NHS)-esters (react with Lys residues) and / or maleimide groups (react with thiol groups). In some embodiments, the cross-linking agent can comprise two different linking chemistries (e.g., NHS-ester on one end and maleimide on the other end). In any of these embodiments, the peptides can be the same or different.

[0211] In some embodiments, TNFi peptides are cross-linked through the use of a multimeric scaffold. The multimeric scaffold can comprise two or more functional groups for linking TNFi peptides, each TNFi peptide optionally linked to the multimeric scaffold through a linker. In some embodiments, each TNFi peptide is linked to a reactive group on the multimeric scaffold through a linker, such as a PEG group. In some embodiments, each TNFi peptide is linked to a reactive group on the multimeric scaffold through a linker, such as a PEG group.

[0212] In some embodiments, the multimeric scaffold can be a dimeric scaffold comprising two functional groups, such as NHS ester groups. In some embodiments, the multimeric scaffold can comprise two identical functional groups, such as NHS ester groups. In some embodiments, the multimeric scaffold can comprise at least two different functional groups, such as an NHS ester group and a maleimide group. In some embodiments, the multimeric scaffold can be a trimeric scaffold comprising three functional groups, such as NHS ester groups or o-pentafluorophenyl ester groups. In some embodiments, the multimeric scaffold can comprise three identical functional groups, such as NHS ester groups or o-pentafluorophenyl ester groups. In some embodiments, the multimeric scaffold can comprise at least two different functional groups, such as an NHS ester group or o-pentafluorophenyl ester group and a maleimide group. In some embodiments, the multimeric scaffold can be a tetrameric scaffold comprising four functional groups, such as three NHS ester groups, or an o-pentafluorophenyl ester group and a fourth orthogonal group. In some embodiments, the multimeric scaffold can comprise different functional groups, such as an NHS ester group, an o-pentafluorophenyl ester group, a maleimide group, and a fourth orthogonal group. In some embodiments, the functional groups can be Fmoc-amine, nitro, amine, or carboxylate.

[0213] In some embodiments, the orthogonal group can be a functional group for linking an auxiliary molecule. In some embodiments, the orthogonal group can be, for example, a PEG chain, a cholesterol moiety, a biotin group, a lipid (fatty acid group), a nitro group, or a protected amine. In some embodiments, the PEG chain of the orthogonal group has 2 to 48 ethylene glycol repeat units, or 2 to 24 ethylene glycol repeat units. In some embodiments, the orthogonal group is a cholesteryl-PEG4-NHS ester, as shown in the following figure, where n = 4:

[0214] In some embodiments, the TNFi peptide is linked to the multimeric scaffold through a linker. In some embodiments, the linker comprises a PEG group with a functional group at its end. In some embodiments, the PEG group has an NHS ester at both ends. In some embodiments, the PEG group has an o-pentafluorophenyl ester at both ends. In some embodiments, the PEG group has an amine group at one end and an NHS ester at the other end. In some embodiments, the PEG group has an amine group at one end and an o-pentafluorophenyl ester at the other end. In some embodiments, the PEG group has a carboxyl group at one end and an o-pentafluorophenyl ester at the other end. In some embodiments, the PEG group has an amine or carboxyl group at one end and an NHS ester or o-pentafluorophenyl ester at the other end. In some embodiments, the PEG group has an amine or carboxyl group at one end and a carboxyl or amine group at the other end.

[0215] In some embodiments, the multimeric scaffold is a tri-functional crosslinker, such as tris(succinimidyl) aminotriacetate (TSAT), which contains three N-hydroxysuccinimide (NHS) ester groups. In some embodiments, the multimeric scaffold is a tri-functional crosslinker, such as tris-succinimidyl (6-aminocaproyl) aminotriacetate (LC-TSAT). In some embodiments, the multimeric scaffold is a heterotetrameric PEG scaffold, with three functional groups of the same type and an additional reactive group that is orthogonal to the other functional groups. In some embodiments, the multimeric scaffold is a heterotetrameric PEG scaffold, with three NHS ester functional groups and an additional maleimide reactive group. In some embodiments, the PEG length of the linker connecting the TNFi peptide to the scaffold is different from the length of the PEG group attached to the orthogonal reactive group.

[0216] In some embodiments, the multimeric scaffold is a Fmoc scaffold (bis(2,5- dioxopyrrolidin-1-yl) 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-((2,5- dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)heptanedioate). In some embodiments, the multimeric scaffold is a Fmoc scaffold with PEG27 chains (see Figure 15A In some embodiments, the multimeric scaffold is a cyclohex scaffold (tris(2,5- dioxopyrrolidin-1-yl)cyclohexane-1,3,5-tricarboxylate). In some embodiments, the multimeric scaffold is a nitro scaffold (bis(2,5-dioxopyrrolidin-1-yl) 4-(3-((2,5- dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-4-nitroheptanedioate). In some embodiments, the multimeric scaffold is an amine scaffold (bis(2,5-dioxopyrrolidin-1-yl) 4-amino-4-(3-((2,5- dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)heptanedioate). In some embodiments, the multimeric scaffold is an amine-PEG27 scaffold, as shown in Figure 16A In some embodiments, the multimeric scaffold is a cholesteryl scaffold, as shown in Figure 16AIn some embodiments, the heterotetrameric PEG scaffold is based on a 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)-methyl]propoxy}propanoic acid scaffold. In some embodiments, the multimeric scaffold is based on 4-amino-4-(2-carboxyethyl) pimelic acid. In some embodiments, the TNFi peptide comprises a C-terminal D-lysine residue that is linked to a carboxyl group of the 4-amino-4-(2-carboxyethyl) pimelic acid. In some embodiments, the TNFi peptide multimer comprises three TNFi peptides, each with a C-terminal D-lysine residue that is linked to a carboxyl group of the 4-amino-4-(2-carboxyethyl) pimelic acid. In some embodiments, such TNFi peptide multimers further comprise a helper molecule linked to a free amino group of a carboxyl group of the 4-amino-4-(2-carboxyethyl) pimelic acid scaffold. In some further embodiments, the helper molecule is a PEG group.

[0217] In some embodiments, the PEG group of the linker to the multimeric scaffold has 1 to 48 subunits. In some embodiments, the PEG group of the linker has 1 to 30 subunits. In some embodiments, the PEG group of the linker has 1 to 24 subunits. In some embodiments, the PEG group of the linker has 1 to 12 subunits. In some embodiments, the PEG group of the linker has 4 to 30 subunits. In some embodiments, the PEG group of the linker has 4 to 24 subunits. In some embodiments, the PEG group of the linker has 4 to 12 subunits. In some embodiments, the PEG group of the linker has 4 to 10 subunits. In some embodiments, the PEG group of the linker has 4 subunits. In some embodiments, the PEG group of the linker has 6 subunits. In some embodiments, the PEG group of the linker has 8 subunits. In some embodiments, the PEG group of the linker has 10 subunits. In some embodiments, the PEG group of the linker has 12 subunits. In some embodiments, the PEG groups of the linkers that are part of the multimer can have the same or different lengths. In some embodiments, the PEG group can be composed of a single PEG chain, or of a first PEG chain and a second PEG chain linked in tandem. In some embodiments, the PEG group can comprise an internal NHS-ester bond or other bond, for example a bond formed by linking multiple PEG groups together.

[0218] In some embodiments, the linker or multimeric scaffold can comprise a tris, di-lysine, benzene ring, phosphate, or peptide core as a functional group or reactive group. In some embodiments, the cross-linking group comprises a bis(sulfosuccinimidyl)suberate (B3S), disuccinimidyl glutarate (DSG), or bis(sulfosuccinimidyl)suberate (DST) group. In some embodiments, the cross-linking group comprises a thiol-reactive group, such as haloacetyl (e.g., iodoacetate), pyridyl disulfide (e.g., HPDP), and others.

[0219] Accessory molecules linked to TNFi peptides or multimers thereof

[0220] In some embodiments, the TNFi peptide or multimer thereof can be modified or linked to an auxiliary molecule, such as a potency-enhancing molecule, a stabilizing molecule, or other molecule that provides an increase in activity, potency, binding, pharmacokinetics, membrane localization, or other property.

[0221] In some embodiments, the multimeric scaffold can comprise an auxiliary molecule, such as a PEG molecule (e.g., linear or branched), a sterol (e.g., cholesterol) or analog thereof (e.g., thiocholesterol), a sugar, a maltose binding protein, a biotin group, a lipid (fatty acid), serum albumin, ubiquitin, streptavidin, an immunoglobulin domain, a keyhole limpet hemocyanin, a sperm whale myoovalbumin, a green fluorescent protein, a gold particle, a magnetic particle, an agarose bead, a lactose bead, an alkane chain (e.g., C8, C1-C6, or C1-C8 alkyl chain, etc.), or a fatty acid (e.g., C8 fatty acid, C 16 fatty acid, palmitate, etc.). In other embodiments, the auxiliary molecule can be a linkage of multiple multimers, such as a linkage of multiple trimers (e.g., to increase molecular weight and decrease kidney filtration). 18 fatty acid, palmitate, etc.). In other embodiments, the auxiliary molecule can be a linkage of multiple multimers, such as a linkage of multiple trimers (e.g., to increase molecular weight and decrease kidney filtration).

[0222] In some embodiments, the TNFi peptide or multimer thereof comprises an auxiliary molecule, such as a label or other detectable marker. In some embodiments, such a label or other detectable marker is linked to the N-terminus and / or C-terminus of the TNFi peptide. In some embodiments, the TNFi peptide is labeled at the N-terminus. In some embodiments, the TNFi peptide is labeled at the N-terminus with a biotin group, for example. In some embodiments, the TNFi peptide is labeled at the C-terminus. In some embodiments, the TNFi peptide is labeled at the C-terminus with a biotin group, for example.

[0223] In some embodiments, the TNFi peptide multimer comprises a label or other detectable marker. In some embodiments, such a label or other detectable marker is attached to the N-terminus and / or C-terminus of the TNFi peptide, or to the orthogonal arm of the multimeric backbone. In some embodiments, the label or other detectable marker is a biotin group.

[0224] In some embodiments, the accessory molecule is attached to the TNFi peptide or multimer thereof via a PEG group. The accessory molecule can be attached to the PEG group using various chemical methods known in the art. For example, the accessory molecule can be attached to the PEG group via a carbamate, which is formed by the reaction of a halocarbonyl ester molecule with an amine. In another example, the accessory molecule can be attached to the PEG group via an amide bond, which is formed by the condensation of a carboxylic acid molecule with an amine. In another example, the accessory molecule can be attached to the PEG group via an amide bond, which is formed by the reaction of a NHS molecule or any other active ester with an amine. In another example, the accessory molecule can be attached to the PEG molecule chain via an amide bond, which is formed by the reaction of a ketone with an amine (isourea). In another example, the accessory molecule can be attached to the PEG chain via a thioether bond, which is formed by the reaction of a thiol with a maleimide ester. In another example, the accessory molecule can be attached to the PEG group via an ether bond, for example by the dehydration reaction of a terminal hydroxyl group on the accessory molecule with a PEG group. In another example, the accessory molecule can be attached to the PEG group via click chemistry, for example the Huisgen 1,3-dipolar cycloaddition reaction between an azide and an alkyne.

[0225] In some embodiments, the PEG group attached to the accessory molecule can be composed of a single PEG chain or a first PEG chain and a second PEG chain connected in tandem. In some embodiments, the PEG group accessory molecule can comprise an internal NHS-ester bond or other bond, for example a bond formed by linking multiple PEG groups together.

[0226] Avidity of multimers

[0227] The TNFi peptide multimer can have an increased affinity for the TNFα molecule compared to the affinity of a single TNFi peptide or control peptide for the TNFα molecule. In some embodiments, the TNFi peptide multimer can have an increased affinity for the trimeric TNFα compared to the affinity of a single TNFi peptide or control peptide for the trimeric TNFα. In some embodiments, the TNFi peptide multimer is a dimer. In some embodiments, the TNFi peptide multimer is a trimer. The single peptide or control peptide can be the same as one of the components of the multimer, or the single peptide can be a different peptide not contained in the multimer.

[0228] Compared to the affinity of the TNFi peptide alone, the affinity of the TNFi peptide multimer for the trimer TNFα can show an increase of approximately 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, or 10000 times.

[0229] Pharmaceutical compositions

[0230] TNFi peptides and their multimers can be administered in vivo as pharmaceutical compositions. Pharmaceutical compositions typically contain pharmaceutically acceptable excipients, carriers, and / or other components. "Pharmaceutically acceptable" means that the components of the composition are not biologically or otherwise undesirable, i.e., these components can be administered to a subject (e.g., a human) together with the TNFi peptide or its multimer without causing any undesirable biological effects or harmful interactions with any other component of the pharmaceutical composition containing it. Pharmaceutically acceptable excipients are selected to minimize degradation of the active ingredient (e.g., the TNFi peptide or its multimer) and to minimize any adverse side effects on the subject, as is well known to those skilled in the art.

[0231] Pharmaceutical compositions can be formulated into any suitable form of administration. They can be formulated for oral administration, parenteral administration (e.g., intravenous, intramuscular, or subcutaneous administration), topical administration (e.g., transdermal), rectal administration, etc. The required dosage of the pharmaceutical composition varies from subject to subject, depending on the subject's species, age, weight and general condition, severity of disease, and route of administration. Those skilled in the art can determine the appropriate dosage using only routine experiments based on the teachings herein.

[0232] Parenteral administration of pharmaceutical compositions is typically characterized by injection. Injectable formulations can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for dissolving in or suspending in a liquid prior to injection, or emulsions. Parenteral administration may also utilize sustained-release or continuous-release systems (i.e., depots) to maintain a constant dose.

[0233] Suitable excipients, carriers, and their formulations are described in Remington: The Science and Practice of Pharmacy (19th Ed.) A. R. Gennaro, ed., Mack Publishing Company, Easton, PA 1995. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution can be from about 5 to about 8, or from about 7 to about 7.5. Other carriers include sustained-release formulations, such as a solid hydrophobic polymer matrix semipermeable matrix containing a TNFi peptide or multimer thereof, which is a shaped article, such as a film, a liposome, or a microparticle. It will be apparent to those skilled in the art that certain carriers can be more preferred, depending, for example, on the route of administration and the concentration of the composition being administered.

[0234] Carriers for inclusion in pharmaceutical compositions are well-known to those skilled in the art. These carriers are generally standard carriers for administering drugs to the human body, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously.

[0235] Pharmaceutical compositions can include, in addition to a TNFi peptide or multimer thereof, carriers, thickening agents, diluents, buffers, preservatives, surface active agents, and the like. The pharmaceutical compositions can also include one or more other active ingredients, such as antibacterial agents, anti-inflammatory agents, anesthetics, and the like.

[0236] Formulations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including, for example, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluids and nutrient supplements, electrolyte supplements such as Ringer's dextrose-based supplements, and the like. Preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, inert gases, and the like can also be added.

[0237] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. Additionally, it is also contemplated herein that compositions designed for oral administration can also include enteric permeation agents.

[0238] In some embodiments, the TNFi peptide or multimer thereof can be administered as a pharmaceutically acceptable acid addition salt or base addition salt formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanol amines.

[0239] In some embodiments, the pharmaceutical composition of the TNFi peptide or multimer thereof is lyophilized. Formulations of the TNFi peptide or multimer thereof can be lyophilized for reconstitution or made into a liquid form. The terms “lyophilization” and “lyophilized” and “freeze-dried” refer to a process by which a material to be dried is first frozen and then subjected to a vacuum environment in which ice or frozen solvent is removed by sublimation. Excipients can be included in the formulation prior to lyophilization to enhance the stability of the lyophilized product upon storage. A “reconstituted” formulation refers to a formulation prepared by dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration (e.g., parenteral administration), and optionally, subcutaneous administration or other routes of administration.

[0240] Uses

[0241] The TNFi peptides and multimers thereof can be used to reduce TNFα-mediated inflammation in a subject. In some embodiments, a TNFi peptide or multimer thereof is administered to reduce TNFα-mediated inflammation in a subject. In some embodiments, a TNFi peptide or multimer thereof is administered to reduce the activity of TNFα in a subject. In some embodiments, a TNFi peptide or multimer thereof is administered to reduce an inflammatory response mediated by TNFα. The TNFi peptide or multimer thereof can be administered by oral administration, parenteral administration, intravenous administration, intramuscular administration, subcutaneous administration, topical administration, rectal administration, and the like. In some embodiments, a TNFi peptide or multimer thereof is administered topically to achieve local suppression of TNFα activity or TNFα-mediated inflammation or TNFα-mediated inflammatory response.

[0242] The TNFi peptides and multimers thereof can be used to treat a subject having a TNFα-mediated disease. In some embodiments, a TNFi peptide or multimer thereof is administered to a subject in need thereof to treat a TNFα-mediated disease. In some embodiments, the TNFα-mediated disease is an inflammatory disease or disorder. In some embodiments, the TNFα-mediated disease is an autoimmune disease.

[0243] In some embodiments, the TNFi peptide or multimer thereof is administered orally. In some embodiments, the TNFi peptide or multimer thereof is administered parenterally. In some embodiments, the TNFi peptide or multimer thereof is administered intravenously. In some embodiments, the TNFi peptide or multimer thereof is administered intramuscularly. In some embodiments, the TNFi peptide or multimer thereof is administered subcutaneously. In some embodiments, the TNFi peptide or multimer thereof is administered topically. In some embodiments, the TNFi peptide or multimer thereof is administered rectally.

[0244] The term "subject" as used herein refers to a human or an animal. Typically, the animal is a vertebrate such as a primate, rodent, domestic animal, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic animals and game animals include cows, horses, pigs, deer, bison, water buffalo, feline animals (e.g., domestic cats), canine animals (e.g., dogs, foxes, wolves), avian animals (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. In some embodiments, the subject is a human, monkey, and dog. In some embodiments, the subject is a human.

[0245] In some embodiments, the TNFi peptide or multimer thereof is administered to a subject in need thereof to treat a TNFα-mediated disease, e.g., adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, cutaneous lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (polyarticular), hidradenitis suppurativa, uveitis (intermediate, posterior, or pan-uveitis), or radiographic negative axial spondyloarthritis.

[0246] In some embodiments, the TNFi peptide or multimer thereof is administered to a subject in need thereof who has an inflammatory bowel disease, e.g., adult or pediatric Crohn's disease or ulcerative colitis. In some embodiments, the TNFi peptide is administered orally to the subject.

[0247] In some embodiments, the TNFi peptide or multimer thereof is administered to a subject in need thereof who has an inflammatory bowel disease, e.g., adult or pediatric Crohn's disease or ulcerative colitis. In some embodiments, the TNFi peptide is administered rectally (e.g., via an enema) to the subject.

[0248] In some embodiments, the TNFi peptide or multimer thereof is administered to a subject in need thereof who has an inflammatory skin disease, e.g., plaque psoriasis, hidradenitis suppurativa, or cutaneous lupus. In some embodiments, the TNFi peptide is administered topically to the subject.

[0249] In some embodiments, the TNFi peptide or multimer thereof is administered to a subject in need thereof having an inflammatory skin disease (e.g., plaque psoriasis, hidradenitis suppurativa, or cutaneous lupus). In some embodiments, the TNFi peptide or multimer thereof is administered parenterally (e.g., subcutaneously, intramuscularly, or intravenously) to the subject. In some embodiments, the TNFi peptide or multimer thereof is administered subcutaneously to the subject. In some embodiments, the TNFi peptide or multimer thereof is administered intramuscularly to the subject. In some embodiments, the TNFi peptide or multimer thereof is administered intravenously to the subject.

[0250] In some embodiments, the TNFi peptide or multimer thereof is administered to a subject in need thereof having an inflammatory disease (e.g., systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis). In some embodiments, the TNFi peptide or multimer thereof is administered parenterally (e.g., subcutaneously, intramuscularly, or intravenously) to the subject. In some embodiments, the TNFi peptide or multimer thereof is administered subcutaneously to the subject. In some embodiments, the TNFi peptide or multimer thereof is administered intramuscularly to the subject. In some embodiments, the TNFi peptide or multimer thereof is administered intravenously to the subject.

[0251] Effective administration doses and administration regimens of the TNFi peptide or multimer thereof can be determined empirically, and such determination is within the ability of one skilled in the art. The administration dose of the TNFi peptide or multimer thereof and compositions thereof should be large enough to produce the desired therapeutic effect on the symptoms / disease. The dose should generally not be so large as to cause serious adverse side effects, such as unwanted cross-reactions, allergic reactions, etc. Generally, the dose will vary according to the age, condition, sex, extent of disease, route of administration, or whether other drugs are included in the regimen, and can be determined by one skilled in the art. The physician can adjust the dose individually if there are any contraindications. The dose can vary and can be administered once or more times daily for one or several days.

[0252] In some embodiments, the typical daily dose of the TNFi peptide or multimer thereof used can range from about 1 pg / kg body weight to up to 100 mg / kg body weight or more per day, depending on the factors mentioned above. In some embodiments, the typical daily dose of the TNFi peptide or multimer thereof used can range from about 0.01 pg / kg body weight to up to 1 mg / kg body weight or more per day. In some embodiments, the administration of the TNFi peptide or multimer thereof can be several times daily, daily, weekly, monthly, or yearly, depending on the condition of the subject, other modes of treatment, etc. A suitable administration regimen can be readily determined by one skilled in the art.

[0253] A TNFi peptide or multimer thereof can be administered prophylactically to a patient or subject at risk of developing a TNFa mediated disease or at risk of recurrence of a TNFa mediated disease.

[0254] Methods of making TNFi peptides and multimers thereof

[0255] A TNFi peptide or multimer thereof can be prepared using any method known to those skilled in the art for preparing D-peptides or multimers thereof. The TNFi peptides can be linked, for example, by disulfide cross-linking. For example, the D-peptides disclosed herein have two Cys residues that are linked by a disulfide bond, which cyclizes the peptide and forms a more compact structured peptide. The disulfide bond is believed to have enhanced TNFa binding activity.

[0256] Two or more TNFi peptides can also be linked together by protein chemistry techniques. For example, the peptides or polypeptides can be chemically synthesized using currently available laboratory equipment employing FMOC (fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistry (Applied Biosystems, Inc., Foster City, CA). Those skilled in the art will readily appreciate that, for example, a peptide corresponding to any of the disclosed TNFi peptides can be synthesized by standard chemical reactions. For example, a TNFi peptide can be synthesized but not cleaved from its synthesis resin, while another peptide fragment can be synthesized and subsequently cleaved from the resin, thereby exposing an end group that is functionally blocked on the other fragment. Through a peptide condensation reaction, the two fragments can be covalently linked through a peptide bond at their carboxyl and amino terminal ends, respectively, forming a TNFi peptide. (Grant GA (1992) Synthetic Peptides: A User Guide. W. H. Freeman and Co., N.Y. (1992); Bodansky M. and Trost B. Ed. (1993) Principles of Peptide Synthesis. Springer Verlag Inc., NY (both of which incorporate materials related to peptide synthesis by reference herein)). Once isolated, these individual peptides can be linked by similar peptide condensation reactions to form a peptide.

[0257] For example, enzymatic ligation of cloned peptide segments or synthetic peptide segments can be used to join relatively short peptide fragments to produce larger peptides (Abrahmsen L., et al., Biochemistry, 30: 4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can also be used to synthesize larger peptides from shorter peptide fragments. This method consists of two chemical reactions (Dawson et al., Synthesis of Proteins by Native Chemical Ligation. Science, 266: 776 779 (1994)). The first step is a chemoselective reaction of an unprotected synthetic peptide thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. This intermediate undergoes a spontaneous, rapid intramolecular reaction without changing the reaction conditions to form a native peptide bond at the ligation site (Baggiolini M. et al., (1992) FEBS Lett. 307: 97-101; Clark-Lewis I. et al., J. Biol. Chem., 269: 16075 (1994); Clark-Lewis I. et al., Biochemistry, 30: 3128 (1991); Rajarathnam K. et al., Biochemistry 33: 6623-6630 (1994)).

[0258] Alternatively, unprotected peptide segments can be joined chemically, where the bond formed between the peptide segments as a result of the chemical ligation is a non-native (non-peptide) bond (Schnolzer, M. et al., Science, 256: 221 (1992)). This technique has been used to synthesize analogs of protein domains as well as relatively pure proteins in large quantities with full biological activity (de Lisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257 267 (1992)).

[0259] The present application is further illustrated by the following embodiments, which should not be construed as limiting. 1. A D-peptide or salt thereof, wherein the peptide comprises a core TNFα binding domain of D-amino acids and has the amino acid sequence: C-X 2 -X 3 -X 4 -X 5 -X 6 -F-N-N-X 10-X 11 -C (SEQ ID NO: 1), wherein X 1 to X 6 , X 10 , and X 11 each is a D-amino acid or a D a-amino acid analog thereof, and C, F, and N are D-cysteine, D-phenylalanine, and D-asparagine or a D a-amino acid analog thereof; and a. X 2 is D-Thr (T), D-Val (V), D-His (H), D-Leu (L), D-Gln (Q), D-Ala (A), D-Ile (I), D-Met (M), or D-Trp (W), or a D a-amino acid analog thereof; b. X 3 is selected from D-Trp (W), D-Phe (F), D-Tyr (Y), D-Ser (S), D-His (H), or D-Leu (L), or a D a-amino acid analog thereof; c. X 4 is a polar amino acid selected from D-Arg (R), D-Lys (K), D-His (H), D-Glu (E), D-Asp (D), D-Gln (Q), D-Asn (N), D-Thr (T), D-Ser (S), D-Pro (P), D-Ala (A), or D-Gly (G), or a D a-amino acid analog thereof; d. X 5 is D-Pro (P), D-Trp (W), D-His (H), D-Gln (Q), D-Arg (R), D-Ala (A), D-Val (V), D-Leu (L), or D-Gly (G), or a D a-amino acid analog thereof; e. X 6 is D-Arg (R), D-His (H), D-Lys (K), D-Glu (E), D-Gln (Q), D-Val (V), D-Leu (L), D-Ser (S), or D-Ala (A), or a D a-amino acid analog thereof; f. X 10 is D-Trp (W) or D-Tyr (Y), or a D a-amino acid analog thereof; g. X 11 is D-Trp (W), D-Tyr (Y), D-Gln (Q), or D-His (H), or a D a-amino acid analog thereof; and h. C represents D-cysteine or a D a-amino acid analog thereof, F represents D-phenylalanine or a D a-amino acid analog thereof; and N represents D-asparagine or a D a-amino acid analog thereof. 2. The D-peptide or salt thereof according to embodiment 1, wherein the core TNFα binding domain has the following amino acid sequence: C-X 2 -[W / F / Y]-X4 -X 5 -X 6 -FNN-[W / Y]-WC (SEQ ID NO: 3). 3. The D-peptide or its salt according to any one of embodiments 1 to 2 further comprises an intramolecular disulfide bond between cysteine ​​residues of the core TNFα binding domain. 4. A D-peptide or a salt thereof according to any one of embodiments 1 to 3, wherein: aX 2 It is D-type Thr, Val, His, Leu, Gln, Ala, Ile, Met or Trp, or their D-α-amino acid analogs; bX 2 It is D-type Thr, Val, His, Leu or Gln, or their D-α-amino acid analogs; cX 2 Selected from D-type Thr, Val, His, and Leu, or their D-α-amino acid analogs; dX 2 Selected from D-type Thr, Val, and His, or their D-α-amino acid analogs; eX 2 Selected from D-type Thr and Val, or their D-α-amino acid analogs; fX 2 It is D-type Thr, or its D-α-amino acid analogue; or gX 2 It is D-type Val, or its D-α-amino acid analogue. 5. A D-peptide or a salt thereof according to any one of embodiments 1 or 3 to 4, wherein: aX 3 Selected from D-type Trp, Phe, Tyr and Ser, or their D-α-amino acid analogs; bX 3 Selected from D-type Trp, Phe, and Tyr, or their D-α-amino acid analogs; cX 2 Selected from D-type Trp and Phe, or their D-α-amino acid analogs; dX 3 It is D-type Trp, or its D-α-amino acid analogue; or eX 3 It is D-type Phe, or its D-α-amino acid analogue. 6. A D-peptide or a salt thereof according to any one of embodiments 1 to 5, wherein: aX 4is D-form Arg, His, Gin, Asn, Lys, Thr, or Ser, or a D-a-amino acid analog thereof; b. X 4 is selected from D-form Arg, His, Gin, and Asn, or a D-a-amino acid analog thereof; c. X 4 is D-form Arg, His, or Gin, or a D-a-amino acid analog thereof; d. X 4 is D-form Arg, Gin, or Asn, or a D-a-amino acid analog thereof; or e. X 4 is D-form Gin, or a D-a-amino acid analog thereof. 7. The D-peptide or salt thereof according to any one of embodiments 1 to 6, wherein: a. X 5 is D-form Pro, Trp, His, Gin, Gly, Arg, Ala, Val, or Leu, or a D-a-amino acid analog thereof; b. X 5 is D-form Pro, Trp, His, Gin, Gly, Arg, or Val, or a D-a-amino acid analog thereof; c. X 5 is D-form Pro, Trp, or His, or a D-a-amino acid analog thereof; d. X 5 is D-form Pro or Trp, or a D-a-amino acid analog thereof; or e. X 5 is D-form Pro, or a D-a-amino acid analog thereof. 8. The D-peptide or salt thereof according to any one of embodiments 1 to 7, wherein: a. X 6 is D-form Arg, His, Lys, Glu, Gin, Val, Leu, Ser, or Ala, or a D-a-amino acid analog thereof; b. X 6 is D-form Arg, His, Lys, Glu, Gin, Val, or Leu, or a D-a-amino acid analog thereof; c. X 6 is D-form Arg, His, Lys, Glu, or Gin, or a D-a-amino acid analog thereof; d. X 6 is D-form Arg, His, Lys, or Glu, or a D-a-amino acid analog thereof; e. X 6 is D-form Arg or His, or a D-a-amino acid analog thereof. f.X 6 is D-Arg, or a D-a-amino acid analog thereof; or g.X 5 is D-His, or a D-a-amino acid analog thereof. 9. The D-peptide or salt thereof according to any one of embodiments 1 and 3 to 8, wherein: a.X 10 is D-Trp, or a D-a-amino acid analog thereof; or b.X 10 is D-Tyr, or a D-a-amino acid analog thereof. 10. The D-peptide or salt thereof according to any one of embodiments 1 and 3 to 9, wherein: a.X 11 is D-Trp (W), Tyr (Y), or Gin (Q), or a D-a-amino acid analog thereof; or b.X 11 is D-Trp (W), or a D-a-amino acid analog thereof. 11. The D-peptide or salt thereof according to any one of embodiments 1 to 10, wherein each of X 1 to X 6 , X 10 , and X 11 is a D-a-amino acid. 12. The D-peptide or salt thereof according to any one of embodiments 1 to 11, wherein the core TNFα binding domain has the following amino acid sequence: a. CVWQPKFNNYWC (SEQ ID NO: 4); b. CVWQPRFNNYWC (SEQ ID NO: 5); c. CTFQPRFNNYWC (SEQ ID NO: 6); d. CTFQPRFNNWWC (SEQ ID NO: 7); e. CSFQPRFNNYWC (SEQ ID NO: 8); f. CSFQPRFNNWWC (SEQ ID NO: 9); g. CVFQPRFNNYWC (SEQ ID NO: 10); h. CVFQPRFNNWWC (SEQ ID NO: 11); i. CTFQWRFNNYWC (SEQ ID NO: 12); j. CLYQPVFNNWWC (SEQ ID NO: 13); k. CVFQAAFNNYWC (SEQ ID NO: 14); l. CVFQHHFNNWWC (SEQ ID NO: 15); m. CHFNPRFNNWWC (SEQ ID NO: 16); n. CVWQPHFNNYWC (SEQ ID NO: 17); o. CVFQGRFNNWWC (SEQ ID NO: 18); p. CVFQHRFNNWWC (SEQ ID NO: 19); q. CVFNPRFNNWWC (SEQ ID NO: 20); r. CVFKPRFNNWWC (SEQ ID NO: 21); s. CAYQRQFNNWWC (SEQ ID NO: 22); t. CWFEHRFNNWHC (SEQ ID NO: 23); u. CHFQHRFNNWWC (SEQ ID NO: 24); v. CHFQPRFNNWWC (SEQ ID NO: 25); w. CTYQPRFNNWWC (SEQ ID NO: 26); x. CQFQPRFNNWQC (SEQ ID NO: 27); or y. CHFSQRFNNWWC (SEQ ID NO: 28). 13. The D-peptide or salt thereof according to any one of the preceding embodiments, wherein the core TNFα binding domain has an amino acid sequence of SEQ ID NOs: 77 to 110. 14. The D-peptide or salt thereof according to any one of the preceding embodiments, further comprising a tag sequence attached to the N-terminus of the peptide. 15. The D-peptide or salt thereof according to embodiment 14, wherein the tag comprises the amino acid sequence D-Asp or D-AspAsp (DD). 16. The D-peptide or salt thereof according to any one of the preceding embodiments, further comprising a tag sequence attached to the C-terminus of the peptide. 17. The D-peptide or salt thereof according to embodiment 16, wherein the tag comprises the amino acid sequence D-GGEEEK (SEQ ID NO: 30) or D-GGRRRK (SEQ ID NO: 31). 18. The D-peptide or salt thereof according to any one of the preceding embodiments, wherein the N-terminus of the peptide comprises a cap. 19. The D-peptide or salt thereof according to embodiment 18, wherein the cap comprises an acetyl group or a protecting group. 20. The D-peptide or salt thereof according to any one of the preceding embodiments, wherein the C-terminus of the peptide comprises a cap. 21. The D-peptide or salt thereof according to embodiment 20, wherein the cap comprises an amide group or a protecting group. 22. The D-peptide or salt thereof according to any one of the preceding embodiments, further comprising a polyethylene glycol (PEG) group. 23. The D-peptide or salt thereof according to any one of the preceding embodiments, further comprising a linker. 24. The D-peptide or salt thereof according to embodiment 23, wherein the linker comprises a PEG group. 25. The D-peptide or salt thereof according to any one of embodiments 22 or 24, wherein the PEG group is attached to the N-terminus of the D-peptide. 26. The D-peptide or salt thereof according to any one of embodiments 22 or 24, wherein the PEG group is attached to the C-terminus of the D-peptide. 27. The D-peptide or salt thereof according to any one of embodiments 22 and 24 to 26, wherein each PEG group is selected from a PEG group having 1 to 48 subunits, 1 to 30 subunits, 1 to 24 subunits, or 1 to 12 subunits. 28. The D-peptide or salt thereof according to embodiment 27, wherein each PEG group is selected from a PEG group having 6 subunits, 8 subunits, 10 subunits, or 12 subunits. 29. A multimer of the D-peptide according to any one of the preceding embodiments, or a salt thereof. 30. The multimer or salt thereof according to embodiment 29, wherein the multimer is a dimer. 31. The multimer or salt thereof according to embodiment 29, wherein the multimer is a trimer. 32. The multimer or salt thereof according to any one of embodiments 29 to 31, further comprising a multimeric backbone of D-peptides, optionally linked via a linker. 33. The multimer or salt thereof according to embodiment 32, wherein the multimeric backbone is trimeric. 34. The multimer or salt thereof according to embodiment 32, wherein the multimeric backbone is tetrameric. 35. The multimer or salt thereof according to any of embodiments 33 or 34, wherein the tri-functional crosslinker is tris(succinimidyl) aminotriacetate (TSAT), tri-succinimidyl (6-aminohexanoyl) aminotriacetate (LC-TSAT), Fmoc backbone (bis(2,5-dioxopyrrolidin-1-yl) 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)heptanedioate, Fmoc backbone with PEG27 chain, cyclohex backbone (tris(2,5-dioxopyrrolidin-1-yl)cyclohexane-1,3,5-tricarboxylate), nitro backbone (bis(2,5-dioxopyrrolidin-1-yl) 4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-4-nitroheptanedioate), amine backbone (bis(2,5-dioxopyrrolidin-1-yl) 4-amino-4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)heptanedioate), amine-PEG27 backbone, cholesterol backbone, isotetrameric PEG backbone based on 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propanoic acid backbone, multimer backbone based on 4-amino-4-(2-carboxyethyl)heptanedioic acid or 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propanoic acid. 36. The multimer or pharmaceutically acceptable salt thereof according to embodiment 35, wherein the multimer has a structure selected from the group consisting of: a. Fmoc-[peptide-PEG12-K-amide]3; b. Fmoc-[peptide-PEG4-K-amide]3; c. Fmoc-[peptide-PEG8-K-amide]3; d. Fmoc-[Ac-K-PEG12-peptide-amide]3; e. [peptide-PEG6-K-amide]3-Fmoc; f. [peptide-PEG12-K-amide]3-PEG27-Fmoc; g. [peptide-PEG12-K-amide]3-Fmoc; h. [peptide-PEG12-K-amide]3-cyclohexane; i. [peptide-PEG12-K-amide]3-nitro; j. [peptide-PEG12-K-amide]3-PEG27-amine; k. [peptide-PEG12-K-amide]3-amine; l. [peptide-PEG12-K-amide]3-PEG27-biotin; and m. [peptide-PEG12-K-amide]3-PEG27-cholesterol; and wherein the peptide is a D-peptide. 37. The D-peptide or salt thereof according to any one of embodiments 1 to 28, having an amino acid sequence selected from at least one of: a. DDCVWQPKFNNYWC (SEQ ID NO: 32); b. DGACVWQPKFNNYWC (SEQ ID NO: 38); c. DDCVWQPRFNNYWC (SEQ ID NO: 39); d. DDCVWQPRFNNYWCGGRRRK (SEQ ID NO: 40); e. DCVWQPKFNNYWC (SEQ ID NO: 56); f. DDDCVWQPRFNNYWC (SEQ ID NO: 60); g. DDCTFQPRFNNWWC (SEQ ID NO: 61); h. DDCVFQPRFNNYWC (SEQ ID NO: 62); i. DDCSFQPRFNNYWC (SEQ ID NO: 63); and j. DDCVFQPRFNNWWC (SEQ ID NO: 64). 38. The multimer or salt thereof according to any one of embodiments 29 to 36, comprising a D-peptide having an amino acid sequence selected from at least one of: a. DDCVWQPKFNNYWC (SEQ ID NO: 32); b. DGACVWQPKFNNYWC (SEQ ID NO: 38); c. DDCVWQPRFNNYWC (SEQ ID NO: 39); d. DDCVWQPRFNNYWCGGRRRK (SEQ ID NO: 40); e. DCVWQPKFNNYWC (SEQ ID NO: 56); f. DDDCVWQPRFNNYWC (SEQ ID NO: 60); g. DDCTFQPRFNNWWC (SEQ ID NO: 61); h. DDCVFQPRFNNYWC (SEQ ID NO: 62); i. DDCSFQPRFNNYWC (SEQ ID NO: 63); and j. DDCVFQPRFNNWWC (SEQ ID NO: 64). 39. A pharmaceutical composition comprising at least one D-peptide or multimer as shown in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier. 40. The pharmaceutical composition according to embodiment 39, which is formulated for parenteral administration. 41. The pharmaceutical composition according to embodiment 40, which is formulated for intravenous, intramuscular, or subcutaneous administration. 42. The pharmaceutical composition according to embodiment 39, which is formulated for oral administration. 43. The pharmaceutical composition according to embodiment 39, which is formulated for topical administration. 44. The pharmaceutical composition according to embodiment 43, which is formulated for topical administration to the skin (dermis) or the eye. 45. The pharmaceutical composition according to embodiment 39, which is formulated for rectal administration. 46. A lyophilized composition comprising at least one D-peptide or multimer according to any of embodiments 1 to 38, or a salt or pharmaceutically acceptable salt thereof, and a stabilizer. 47. The lyophilized composition of a pharmaceutical composition according to any of embodiments 39 to 45 and a stabilizer. 48. A rehydrated solution of the lyophilized composition according to embodiment 46 or 47. 49. A method of treating a TNFa-mediated disease comprising administering an effective amount of a D-peptide according to any of embodiments 1 to 28 or 37, a multimer according to any of embodiments 29 to 36 and 38, or a pharmaceutical composition according to any of embodiments 39 to 45, or a pharmaceutically acceptable salt thereof. 50. The method according to embodiment 49, wherein the TNFa-mediated disease is adult Crohn’s disease, pediatric Crohn’s disease, ulcerative colitis, plaque psoriasis, cutaneous lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (polyarticular), hidradenitis suppurativa, uveitis (intermediate, posterior, or pan-uveitis), or radiographic negative axial spondyloarthritis. 51. The method according to embodiment 49, wherein the TNFa-mediated disease is an inflammatory bowel disease. 52. The method according to embodiment 51, wherein the inflammatory bowel disease is adult Crohn’s disease, pediatric Crohn’s disease, or ulcerative colitis. 53. The method according to implementation plan 51 or 52, wherein the administration is oral. 54. The method according to implementation plan 51 or 52, wherein the application is rectal. 55. The method according to implementation plan 51 or 529, wherein the application is parenteral. 56. According to the method of implementation scheme 49, the TNFα-mediated disease is an inflammatory skin disease. 57. According to the method of implementation plan 56, the inflammatory skin disease is plaque psoriasis, hidradenitis suppurativa, or cutaneous lupus. 58. The method according to implementation plan 56 or 57, wherein the application is a local application. 59. The method according to implementation plan 56 or 57, wherein the application is parenteral. 60. According to the method of implementation scheme 49, wherein the TNFα-mediated disease is an inflammatory disease, and wherein the inflammatory disease is systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis or juvenile idiopathic arthritis (polyarticular type). 61. The method according to implementation plan 60, wherein the application is parenteral. 62. The method according to implementation plan 65, 59 or 61, wherein parenteral administration is selected from intravenous, subcutaneous and intramuscular administration. 63. A method for reducing TNFα-mediated inflammation, comprising administering to a subject a D-peptide or its multimer or a pharmaceutically acceptable salt thereof from any of embodiments 1 to 38, or a pharmaceutical composition from any of embodiments 39 to 45. 64. A method for inhibiting TNFα, comprising administering to a subject a D-peptide or polymer thereof or a pharmaceutically acceptable salt thereof from any of embodiments 1 to 38, or a pharmaceutical composition from any of embodiments 39 to 45. 65. A method for reducing TNFα-mediated inflammatory responses, comprising administering to a subject a D-peptide or its multimer or a pharmaceutically acceptable salt thereof from any of embodiments 1 to 38, or a pharmaceutical composition from any of embodiments 39 to 45. 66. The method according to any one of embodiments 63 to 65, wherein the application is by oral administration, parenteral administration, topical (dermal) administration, or rectal administration. 67. The method according to any one of embodiments 63 to 65, wherein the D-peptide or its polymer or a pharmaceutically acceptable salt thereof is applied topically to reduce TNFα activity or inflammation or inflammatory response. 68. The method according to any one of implementation schemes 63 to 67, wherein the subject suffers from a TNFα-mediated disease. 69. The method according to embodiment 68, wherein the TNF alpha mediated disease is adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, cutaneous lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (polyarticular), hidradenitis suppurativa, uveitis (intermediate, posterior, or pan-uveitis), or radiographic negative axial spondyloarthritis. 70. A D-peptide according to any one of embodiments 1 to 38, or a multimer thereof, or a pharmaceutically acceptable salt thereof, for use as a medicament. 71. A D-peptide according to any one of embodiments 1 to 38, or a multimer thereof, or a pharmaceutically acceptable salt thereof, for use in a method of treating a subject by therapy. 72. The D-peptide according to embodiment 71, wherein the subject has a TNF alpha mediated disease. 73. The D-peptide according to embodiment 72, wherein the TNF alpha mediated disease is adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, plaque psoriasis, cutaneous lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis (polyarticular), hidradenitis suppurativa, uveitis (intermediate, posterior, or pan-uveitis), or radiographic negative axial spondyloarthritis.

[0260] The description of embodiments of the present disclosure is not intended to be exhaustive or to be to limit the present disclosure to the precise forms disclosed. While specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the relevant art will recognize. The teachings of the present disclosure provided herein can be applied to other procedures or methods. The various embodiments described herein can be combined to provide further embodiments. The various aspects of the present disclosure can be modified if necessary to employ the compositions, functions, and concepts described above to provide yet further embodiments of the present disclosure. These and other modifications can be made to the disclosure in light of the detailed description.

[0261] The particular elements of any of the foregoing embodiments can be combined with or substituted for those of other embodiments. Moreover, although sets of features have been described above as being associated with certain embodiments, other embodiments can exhibit such features in combination with other embodiments. Not all embodiments necessarily exhibit the same advantages described above.

[0262] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, methodologies described in such publications that might be used in connection with the present disclosure. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as a representation or admission that the inventors are not entitled to use the material of such publications as prior art. All statements as to the date or representations made by others in connection with the publications are based on the information available to the applicants and do not constitute any admission as to the correctness or correctness of the dates or contents of these publications. EMBODIMENTS

[0263] Example 1: Phage display library screening for TNFα antagonistic peptides

[0264] To identify D-peptides that bind to and inhibit the activity of human TNFα, a mirror phage display library was screened using an enantiomeric screening technique. A synthetic, biotinylated D- amino acid human TNFα (i.e., the mirror image of the natural L-amino acid) was used to identify L- amino acid peptides (L-peptides) that specifically bind to the D-amino acid human TNFα. The sequences of L-peptides that bind to the D-amino acid human TNFα amino acids ("hits") were determined by deep sequencing and Sanger sequencing. Based on the sequencing results, D-amino acid peptides of selected L-peptide hits were synthesized and screened for their ability to bind to and inhibit the activity of the natural human TNFα (L-amino acid).

[0265] Synthesis of L-form TNFα

[0266] L-type biotinylated human TNFα was synthesized, which showed similar stability and activity as the natural TNFα protein. The human TNFα protein has the following amino acid sequence: VRSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLI YSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO: 29); corresponding to reference sequence NP_000585.2.

[0267] Chemical methods based on established solid phase peptide synthesis (e.g., Merrifield et al., 1963, J. Am. Chem. Soc. 85:2149-2154; Merrifield, 1986, Science 232:341-347) and native chemical ligation methods (e.g., Dawson et al., 2000, Annu. Rev. Biochem. 69:923-960; Dawson et al., 1994, Science 266:776-779) were used to synthesize L-form human TNFα. Each synthetic intermediate was purified to homogeneity by reverse phase HPLC and characterized by HPLC and LCMS analysis. The final linear protein target was refolded by first forming the individual intramolecular disulfide bonds and then step dialysis from 6 M, 2 M, 0.5 M to 0 M guanidine hydrochloride buffer. The folded biotinylated L-TNFα trimer was separated by SEC (size exclusion chromatography) to remove any aggregated or misfolded material (i.e., to separate the trimer from monomer or higher order aggregates). Synthetic and recombinant L-TNFα proteins were further characterized by biophysical (LC and ESI-MS), biochemical (multiple TNFR binding assays) and activity assays (L-929 cell killing assay) to ensure they were equivalent (data not shown).

[0268] Synthesis of D-form TNFα

[0269] Biotinylated D-TNFα proteins for mirror phage display screening were synthesized using D-amino acids. The folded conformation of the synthesized D-form TNFα proteins was confirmed by electrospray mass spectrometry. The folding was confirmed by a difference of -2 Da due to the formation of one disulfide bond and different ionization patterns. The D-proteins were purified by size exclusion chromatography and the trimeric form was used for subsequent studies.

[0270] Mirror phage display selection process

[0271] Folded biotinylated D-TNFα trimer proteins were subjected to mirror phage display by screening phage libraries of L-peptides with 9 different geometric configurations (1 linear and 8 disulfide bond constrained). The L-peptide libraries were as follows: a linear random 12 amino acid L-peptide library (X 12 ), where each X is any L-amino acid except cysteine; a L-peptide library with the sequence X2CX8CX2, where C is cysteine and each X is any amino acid except cysteine, the subscript numbers indicate the number of L amino acids in each segment of the peptide; a L-peptide library with the sequence CX 10L-peptide libraries of C, where C is cysteine, each X is any L-amino acid except cysteine, and the subscript number indicates the number of L-amino acids between cysteine residues; and six-strand lariat L-peptide libraries of the general formula CX n C, where C is cysteine, each X is any L-amino acid, and n is the number of L-amino acids between cysteine residues (n = 2, 4, 6, 8, 10, 12). The total unique sequence diversity is approximately -4 x 10 10 .

[0272] Next generation deep sequencing and Sanger sequencing of phage binding to biotinylated D-TNFa trimer protein was performed to identify "matchers" (the highest affinity binders). Bioinformatic analysis of the sequences identified >10 different consensus sequences (families of related peptides with certain common residues) (data not shown). Multiple matchers from each family were tested in a phage ELISA assay to identify the best binders to biotinylated D-TNFa trimer protein. Twenty-eight (28) of the identified matchers were further validated by a quantitative assay of the binding of different phage clones to biotinylated D-TNFa protein. Priorities of multiple matchers in each family were determined based on abundance and strength of consensus sequences, synthesized as D-peptides, and tested for binding to L-TNFa and blocking L-TNFa binding to its receptor, as described below. Twenty-eight of the identified phage were further validated by a quantitative assay of the binding of different phage clones to biotinylated D-TNFa protein. Priorities of multiple matchers in each family were determined based on abundance and strength of consensus sequences, synthesized as D-peptides, and tested for binding to L-TNFa protein and blocking L-TNFa binding to its receptor, as described below. The peptides identified by sequencing included peptides with a core TNFa binding domain shown in the following examples and in SEQ ID NOS: 78-111.

[0273] Identification of initial D-peptide binders of TNFα

[0274] The top 28 matchers in the phage display library screening were synthesized as biotinylated D-peptides (Biotin-(D-AspAsp-(D-peptide)-amide) and tested for binding to recombinant human L-TNFa using a sequential solid phase ELISA binding assay, in which the D-peptides were captured to Neutravidin coated plates. Soluble His6-tagged TNFa was detected using an anti-His-HRP secondary antibody. See Figure 1TNFα-018 was the primary match due to its binding to recombinant TNFα (L-form) and its high aqueous solubility. Another match, TNFα-001, was not soluble in aqueous buffer, although it showed strong binding to TNFα according to phage display.

[0275] Binding of D-peptide matches to TNFα was verified in three different ELISA formats: "solid phase" (as described above), "solution phase" (all components mixed in solution and then captured to a Neutravidin surface), and "sequential solid phase" (biotinylated D-peptide mixed with TNFα in solution, captured to a Neutravidin surface, and then secondary antibody added). Binding of D-peptide matches to L-TNFα was also confirmed using a direct fluorescent readout assay (fluorescently labeled D-peptide was used to treat immobilized recombinant biotinylated TNFα). These same fluorescent peptides were also used to record TNFα binding under native conditions by co-elution with recombinant TNFα (L-form) on an analytical size exclusion column (SEC). As described below, selected D-peptide point mutants eliminated binding in these assays. A thermal shift dye binding assay using SYPRO Orange dye (Lavinder et al., 2009, J. Am. Chem. Soc. 131 :3794-3795) was also used to confirm binding. Results indicated that D-peptide (particularly TNF-018α) interaction with TNFα increased its thermal stability (Tm). In addition, SPR (surface plasmon resonance) indicated that immobilized TNFα bound to the original TNFα-018 D-peptide. TM Orange dye binding assay (Lavinder et al., 2009, J. Am. Chem. Soc. 131 :3794-3795) was also used to confirm binding. Results indicated that D-peptide (particularly TNF-018α) interaction with TNFα increased its thermal stability (Tm). In addition, SPR (surface plasmon resonance) indicated that immobilized TNFα bound to the original TNFα-018 D-peptide.

[0276] Example 2: In vitro binding of peptide 18 to TNFα

[0277] Peptide 18 (also known as TNFα-018 and peptide 018) and its variants described below were tested for binding to human TNFα protein (L-form) using a sequential solid phase binding assay. A BSA control was also included in the assay.

[0278] Peptide 18 contains the core TNFa binding domain CVWQPKFNNYWC (SEQ ID NO:4) with a N-terminal biotin molecule attached to a D-AspAsp (DD) dipeptide tag attached to the N-terminus of the peptide core TNFa binding domain; this dipeptide tag improves the solubility of the peptide. Peptide 18 also contains a C-terminal amide group. Peptides 18-V1 and 18-V2 contain the same core TNFa binding domain with the N-terminus acetylated and contain a C-terminal GGEEEK (SEQ ID NO:30) or GGRRRK (SEQ ID NO:31) peptide and biotin-amide group, respectively. These C-terminal sequences were included in peptides 18-V1 and 18-V2 to determine if they improved solubility and / or binding. Peptide 18 mutants were made that were similar in structure to peptide 18 but with the W in the penultimate residue of the C-terminus of the core TNFa binding domain replaced with a G. The peptide sequences are as follows, with differences in the amino acid sequence from peptide 18 underlined: Peptide biotin DD-18: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO:32) Peptide 18-V1 : Ac-CVWQPKFNNYWC GGEEEK (biotin)-amide (SEQ ID NO:33) Peptide 18-V2: Ac-CVWQPKFNNYWC GGRRRK (biotin)-amide (SEQ ID NO:34) Peptide biotin DD-18 (mutant): Biotin-DDCVWQPKFNNY G C-amide (SEQ ID NO:35)

[0279] In the assay, the peptides were immobilized on neutravidin plates and washed with wash buffer (PBS with 0.1% BSA and 0.01% Tween-20, pH 7.4). His6-TEV-TNFa was then added and washed. The TNFa used in the assay was recombinantly expressed human TNFa with a N-terminal His6 tag. The amount of TNFa bound to the peptides was then measured using an anti-His6 tag antibody conjugated to horseradish peroxidase (HRP) and then detected using the QuantaBlu fluorogenic peroxidase substrate (Thermo Scientific). TM

[0280] See Figure 2 ​peptide 18, 18-V1 and 18-V2 showed significant binding to TNFa by this capture assay. For peptide 18 mutants, substitution of Trp (W) to Gly (G) adjacent to the C-terminal Cys residue of the core TNFa binding domain abolished the binding of the peptide to TNFa. The BSA control also showed negligible binding to TNFa. In Figure 2 the number above each bar indicates the fold increase in binding of the peptide to TNFa compared to the BSA control. The amount of binding of the peptides to TNFa in this assay was dependent in part on the relative position and composition of the peptide tag and the position of the biotin molecule.

[0281] Example 3: Peptide 18 blocks TNFa binding to TNFR1

[0282] Peptide biotin DD-18 (Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32)) was tested in a binding assay that measures the disruption of binding between TNFa and one of its receptors, TNFR1 (tumor necrosis factor receptor 1). In this assay, the soluble extracellular domain of TNFR1 with a C-terminal biotin tag was immobilized to neutravidin plates. In parallel, recombinant TNFa with an N-terminal His6 tag (as described in Example 2) was pre-incubated with peptide 18 at concentrations of 10, 25 and 50 micromolar. After pre-incubation, the TNFa-peptide 18 mixture was added to the immobilized TNFR1. BSA protein was used as a negative control. Recombinant expressed llama anti-TNFa nanobody VHH2 (Beirnaert et al., 2017, Front. Immunol. 8:867) was used as a positive control.

[0283] Referring to Figure 3 peptide 18 showed concentration-dependent blocking of TNFa-TNFR1 binding. The BSA protein did not interfere with TNFa-TNFR1 binding, while the positive control anti-TNFa nanobody VHH2 blocked TNFa-TNFR1 binding. In Figure 3 the percentage above each bar indicates the percent reduction in TNFa binding to TNFR1 compared to the negative control. This study demonstrates that peptide 18 is able to bind to TNFa and block its binding to TNFR1.

[0284] Example 4: Peptide 18 blocks TNFa cellular activity on L-929 cells

[0285] The ability of peptide 18 to block TNFα-mediated cell killing was demonstrated using an L-929 cell killing assay (Trost and Lemasters, 1994, Anal. Biochem. 220(1): 149-153). In the presence of actinomycin D, TNFα causes TNFα-TNFR-mediated cell death in this cell line. The sequence of peptide 18 is: Ac-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32; peptide DD-18).

[0286] Cell death was measured by absorbance at 450 nm. Media alone (no TNFα) was the negative control. TNFα plus BSA (no peptide 18) was the positive control.

[0287] See Figure 4A TNFα-induced L-929 cell killing (compare left-most and second columns). Addition of 50 micromolar peptide 18 blocked TNFα-induced cell death (compare second and third columns from left). Reduction of TNFα-induced cell killing was comparable to soluble TNFR1 (2.5 micrograms / milliliter), while anti-TNFα VHH2 nanobody (30 micrograms / milliliter) and anti-TNFα monoclonal antibody (research grade Adalimumab; R&D Systems) showed greater inhibition of TNFα-induced L-929 cell killing.

[0288] In this assay, the concentration of peptide 18 was also varied from 50 micromolar to 0.78125 micromolar, and cell killing was measured as described above (by absorbance at 450 nm). See Figure 4B The left bar in each triplet shows the results for peptide 18 (called B-D-P18; peptide biotin DD-18; SEQ ID NO: 32) alone at the concentration shown, the middle bar shows the results for addition of recombinant human TNFα at a concentration of 0.5 nM and peptide 18 (called B-D-P18; peptide biotin DD-18; SEQ ID NO: 32) at the amount shown, and the right bar shows the results for addition of TNFα at a concentration of 0.1 nM and peptide 18 at the amount shown. (Note: the left-most bar is not present in triplets 8-9 (rTNFα only, and VHH2) because there is no peptide 18.)

[0289] In general, the amount of TNFα-induced cell killing increased as the amount of peptide 18 was decreased. The condition containing control TNFα alone (rTNFα alone) resulted in cell killing as expected. TNFα plus anti-TNFα VHH2 nanobody (VHH2) blocked TNFα-induced cell killing. When peptide DD-18 was replaced with the peptide DD-18 mutant (Biotin-DDCVWQPKFNNYGC-Amide (SEQ ID NO: 35)), TNFα-mediated cell death was not inhibited (18(M) 50 micromolar). The control containing media alone (media) produced the expected results.

[0290] In this study, the EC50values for peptide 18 were calculated to be 9.6 micromolar and 6.1 micromolar under 0.5 nM TNFα and 0.1 nM TNFα conditions, respectively. 50

[0291] Example 5: Comparison of blocking activity of peptide 18 with other primary peptides in the L-929 cell killing assay

[0292] Additional peptide variants of peptide 18 that inhibit TNFα-induced cell death were screened in the L-929 cell killing assay described in Example 4. The peptides screened were peptide 18 (Biotin-DDCVWQPKFNNYWC-Amide; Peptide Biotin DD-18; SEQ ID NO: 32), peptide 18 mutant (Biotin-DDCVWQPKFNNY G C-Amide; SEQ ID NO: 35; Peptide Biotin DD-18 Mutant), and a second mutant, peptide 18 double mutant (Biotin-DDC D WQ D KFNNYWC-Amide; SEQ ID NO: 36; Peptide Biotin DD-18 Double Mutant). The differences between peptide 18 and peptide 18 mutant and peptide 18 double mutant are underlined in the sequence of peptide 18 mutant and peptide 18 double mutant.

[0293] Figure 5 The results are shown. In each pair of bars, the left bar shows the results for the peptide alone. The right bar shows the results for the peptide plus 0.5 mM recombinant human TNFα. Neither peptide 18 mutant nor peptide 18 double mutant showed significant inhibition of TNFα-induced cell killing compared to peptide 18.

[0294] ​The assay tests a trimeric form of peptide 18, peptide 18-IZ (CVWQPKFNNYWC-PEG12-IKKEIEAIKKEQEAIKKKIEAIEKEA-hydrazide; comprising peptide 18 (SEQ ID NO: 4) plus PEG12 plus IZ coiled coil (SEQ ID NO: 46) ). This peptide induces the natural formation of a trimeric IZ coiled coil sequence that forms a trimer of peptide 18 in solution. See again Figure 5 In this assay, the trimeric peptide 18-IZ shows less inhibition than monomeric peptide 18.

[0295] Example 6: Modification of peptide 18 with peptide tags

[0296] In peptide 18 (Biotin-DDCVWQPKFNNYWC-amide; SEQ ID NO: 32; peptide BiotinDD-18), an N-terminal DD dipeptide tag is added to the core TNFα binding domain (CVWQPKFNNYWC; SEQ ID NO: 4) to increase the solubility of the peptide. In Example 2, two additional variants of peptide 18 with C-terminal tags (GGEEEK (SEQ ID NO: 30) and GGRRRK (SEQ ID NO: 31)), peptide 18-V1 (SEQ ID NO: 33) and peptide 18-V2 (SEQ ID NO: 34), were tested and were shown to retain TNFα binding activity. To determine whether the tags affect the binding activity of the core TNFα binding domain to TNFα, peptide 18-V1 (referred to as peptide 18-E3 (Ac-CVWQPKFNNYWC GGEEEK GGRRRK-amide; SEQ ID NO: 34) were tested in the L-929 cell killing assay described in Example 4. (Differences between peptide 18 and the variants are underlined in the variant sequences.) In addition, peptide BiotinDD-18-ex (Biotin- DDCVWQPKFNNYWC GGRRRK GGRRRK-amide; SEQ ID NO: 34) were tested in the L-929 cell killing assay described in Example 4. (Differences between peptide 18 and the variants are underlined in the variant sequences.) In addition, peptide BiotinDD-18-ex (Biotin- DDCVWQPKFNNYWC DD GGRRRK-amide; SEQ ID NO: 34) were tested in the L-929 cell killing assay described in Example 4. (Differences between peptide 18 and the variants are underlined in the variant sequences.) In addition, peptide BiotinDD-18-ex (Biotin- DDCVWQPKFNNYWC GGGSAETVE GGRRRK-amide; SEQ ID NO: 34) were tested in the L-929 cell killing assay described in Example 4. (Differences between peptide 18 and the variants are underlined in the variant sequences.) In addition, peptide BiotinDD-18-ex (Biotin- DDCVWQPKFNNYWC

[0297] See again Figure 5 GGRRRK-amide; SEQ ID NO: 34) were tested in the L-929 cell killing assay described in Example 4. (Differences between peptide 18 and the variants are underlined in the variant sequences.) In addition, peptide BiotinDD-18-ex (Biotin- DDCVWQPKFNNYWC GGRRRK-amide; SEQ ID NO: 34) were tested in the L-929 cell killing assay described in Example 4. (Differences between peptide 18 and the variants are underlined in the variant sequences.) In addition, peptide BiotinDD-18-ex (Biotin- DDCVWQPKFNNYWC

[0298] Example 7: Comparison of binding of other peptide 18 sequence variants in the TNFa binding assay

[0299] The binding of peptide 18 was compared to other sequence variants using the TNFa binding assay described above (Example 2). In addition, another peptide hit from the same CX 10 library, hit 16, was also tested. The following peptides were tested, with the sequence differences from peptide 18 underlined. Peptide 18: Biotin-DD-18-Biotin-DD-CVWQPKFNNYWC-amide (SEQ ID NO: 32) Biotin-DGA-18: Biotin-D GA CVWQPKFNNYWC-amide (SEQ ID NO: 38) Biotin-DD-18-KtoR: Biotin-DDCVWQP R FNNYWC-amide (SEQ ID NO: 39) Biotin-DD-18-KtoR-R3: Biotin-DDCVWQP R FNNYWC GGRRRK -amide (SEQ ID NO: 40) Biotin2-DD-18-KtoR-R3: Biotin-DDCVWQP R FNNYWC GGRRRK (Biotin)-amide (SEQ ID NO: 40) Biotin-DD-hit16: Biotin-DDCHFNP R FNN W WC-amide (SEQ ID NO: 41)

[0300] The results of these binding assays are shown in Figure 6. First, the binding of two different preparations of peptide 18 (Biotin-DD-18; SEQ ID NO: 32) was compared (first and second columns from left). Both preparations of peptide showed comparable binding activity. Figure 6

[0301] Comparing peptide 18 (Biotin-DD-18; SEQ ID NO: 32) to the DGA N-terminal variant (Biotin-DGA-18; SEQ ID NO: 38), it was found that peptide 18 showed better binding (first three columns from left), indicating that the DD peptide tag is superior to the DGA peptide tag.

[0302] ​Comparing peptide 18 (Biotin-DD-18; SEQ ID NO: 32) to a variant of peptide 18 with a Lys to Arg substitution (KtoR) in the core TNFa binding domain (Biotin-DD-18-KtoR; SEQ ID NO: 39) found that the substitution of Lys to Arg enhanced binding (compare left two columns to the fourth column).

[0303] Comparing the binding of peptide Biotin-DD-18-KtoR (with DD N-terminal tag; SEQ ID NO: 39) to peptide Biotin-DD-18-KtoR-R3 (SEQ ID NO: 40) found that the addition of C-terminal GGRRRK (SEQ ID NO: 31) appeared to further enhance binding. The addition of a second biotin molecule to peptide Biotin-DD-18-KtoR-R3 (SEQ ID NO: 40) to form peptide Biotin2-DD-18-KtoR-R3 (SEQ ID NO: 41) appeared to not significantly affect binding. Hit 16 (SEQ ID NO: 42) bound similarly to peptide 18.

[0304] Example 8: Comparison of other peptide 18 sequence variants in the TNFa-TNFR blocking assay

[0305] The blocking ability of peptide 18 was compared to other peptide 18 sequence variants using the TNFa / TNFR blocking assay described above (Example 3). Hit 16 from the same library was also tested. The peptides tested are shown below. Sequence differences from peptide 18 are underlined. In addition, some non-biotinylated peptides were also tested in this study. DD-18: DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) Biotin-DD-18: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) Biotin-DD-18-ex: Biotin-DDCVWQPKFNNYWC GGGSAETVE -amide (SEQ ID NO: 37) Biotin-DGA-18: Biotin- DGA CVWQPKFNNYWC-amide (SEQ ID NO: 38) DGA-18: DGA CVWQPKFNNYWC-amide (SEQ ID NO: 38) DD-18-KtoR: DDCVWQP R FNNYWC-amide (SEQ ID NO: 39) Biotin-DD-18-KtoR: Biotin-DDCVWQP R FNNYWC-amide (SEQ ID NO: 39) Biotin-DD-18-KtoR-R3: Biotin-DDCVWQP R FNNYWC GGRRRK -amide (SEQ ID NO: 40) Biotin2-DD-18-KtoR-R3: Biotin-DDCVWQP R FNNYWCG GRRRK (Biotin)-amide (SEQ ID NO: 40) Biotin-DD-Hit16: Biotin-DDC HFN P R FNN W WC-amide (SEQ ID NO: 41) DD-Hit16: DDC HFN P R FNN W WC-amide (SEQ ID NO: 41)

[0306] See Figure 7 The percentage above each bar represents the percentage of peptide-dependent blockade of TNFa-TNFRl binding. First, two different preparations of peptide 18 (Biotin-DD-18) were tested (Example 7), both of which exhibited similar blocking activity (compare second and third bars from left).

[0307] In this blocking assay, both the non-biotinylated peptide 18 (DD-18) and the biotinylated peptide 18 (Biotin-DD-18) exhibited better blocking than peptide Biotin-DD-18-ex (compare four columns from left). Changing the N-terminal tag of peptide 18 from DD to DGA did not significantly affect the blockade of TNFa-TNFRl binding (compare second through sixth columns from left). The presence or absence of biotin on the N-terminal DGA variant did not appear to significantly affect the blocking of this peptide.

[0308] When the sequence of the core TNFa binding domain was modified by a Lys to Arg substitution (DD-18-KtoR and Biotin-DD-18-KtoR; SEQ ID NO: 39), the blocking ability of these peptides was improved compared to peptide Biotin-DD-18 (compare second and third columns from left to eleventh and twelfth columns).

[0309] When the C-terminal tag (GGRRRK (SEQ ID NO: 31)) was added to the core modified peptide, the blocking activity of the peptide was reduced whether with or without additional biotin molecules (Biotin-DD-18-KtoR-R3; SEQ ID NO: 40 and Biotin2-DD-18-KtoR-R3; SEQ ID NO: 40) (compare columns 11-14 from left).

[0310] Hit 16 with DD N-terminal addition (DDCHFNPRFNNWWC-amide (SEQ ID NO: 41), whether with or without additional biotin molecules (DD-Hit 16 and Biotin-DD-Hit 16), exhibited comparable blocking to the peptide DD-18; SEQ ID NO: 32 (compare columns 4 from left and 15-16).

[0311] In summary, these results indicate that biotin does not contribute to the blocking ability of the peptides, and that substitution of Lys to Arg in the core TNFa binding domain increases the association and blocking of the peptides.

[0312] Example 9: Comparison of other peptide 18 sequence variants in L-929 cell killing assay

[0313] The ability of peptide 18 (designated DD-18; SEQ ID NO: 32) and other sequence variants to block TNFa-induced cell killing was compared using the L-929 cell killing assay described above (Example 4). In addition, biotinylated and non-biotinylated versions of Hit 16 were included. The peptides tested are shown below. Differences in the amino acid sequence from peptide 18 are underlined. Some of the peptides were not biotinylated in this study. DD-18: DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) Biotin-DD-18: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) Biotin-DD-18-ex: Biotin-DDCVWQPKFNNYWC GGGSAETVE -amide (SEQ ID NO: 37) Biotin-DGA-18: Biotin-D GA CVWQPKFNNYWC-amide (SEQ ID NO: 38) DGA-18: D GA CVWQPKFNNYWC-amide (SEQ ID NO: 38) DD-18-KtoR: DDCVWQP RFNNYWC-amide (SEQ ID NO: 39) Biotin-DD-18-KtoR: Biotin-DDCVWQP R FNNYWC-amide (SEQ ID NO: 39) Biotin-DD-18-KtoR-R3: Biotin-DDCVWQP R FNNYWC GGRRRK- amide (SEQ ID NO: 40) Biotin2-DD-18-KtoR-R3: Biotin-DDCVWQP R FNNYWCG GRRRK (Biotin)-amide (SEQ ID NO: 40) DD-hit16: DDC HFN P R FNN W WC-amide (SEQ ID NO: 41) Biotin-DD-hit16: Biotin-DDC HFN P R FNN W WC-amide (SEQ ID NO: 41)

[0314] See Figure 8 First, two different preparations of peptide 18 (Biotin-DD-18; SEQ ID NO: 32) were tested, and both peptide preparations exhibited comparable blocking activity, although with slight differences (compare second and third bars from left). Peptide 18 without the biotin tag (DD-18) blocked cell killing more than peptide 18 with biotin (Biotin-DD-18) (compare second, third, and fourth columns from left). Similarly, peptide 18 without biotin (DD-18) blocked cell killing more than peptide Biotin-DD-18-ex (SEQ ID NO: 37) (compare first and fourth columns from left).

[0315] Changing the N-terminal tag of peptide 18 from DD to DGA, with or without biotin (Biotin-DGA-18 and DGA-18; SEQ ID NO: 38), reduced the ability of the peptides to block cell killing in this assay (compare column 4 with columns 5 and 6). This result is consistent with the results of these peptides in the TNFa binding and TNFa / TNFR assays in Examples 7 and 8 (see above).

[0316] The substitution of Lys to Arg in the core TNFα-binding domain of peptide 18, with or without biotin (Biotin-DD-18-KtoR and DD-18-KtoR; SEQ ID NO: 39), enhanced the peptide's ability to block cell death (compare columns 4 with columns 7 and 8). This result is consistent with the results obtained for these peptides in TNFα binding and TNFα / TNFR assays in Examples 7 and 8 above.

[0317] When a C-terminal tag (GGRRRK (SEQ ID NO: 31)) was added to the core modified peptide, the ability of the peptide to block cell killing was reduced, regardless of whether it had additional biotin molecules (biotin-DD-18-KtoR-R3; SEQ ID NO: 40 and biotin-2-DD-18-KtoR-R3; SEQ ID NO: 41) (compare columns 7 to 10). This result is consistent with the results of these peptides in TNFα binding and TNFα / TNFR assays in Examples 7 and 8 (above).

[0318] Hit 16 with the N-terminal addition of DD (DDCHFNPRFNNWWC-amide (SEQ ID NO: 41), whether or not it has an additional biotin molecule (DD-Hit16 and biotin-DD-Hit16), exhibits blocking activity comparable to that of peptide DD-18 and peptide DD-18 (SEQ ID NO: 32) (comparing columns 2, 3 and 4 from the left with columns 11 and 12).

[0319] In summary, the results of Examples 7 to 9 indicate that modification of the core TNFα-binding domain by Lys-Arg substitution improves peptide activity. The addition of N-terminal and C-terminal peptide tags modulates peptide activity. Biotinylation has minimal effect on peptide activity.

[0320] Example 10: EC of peptide 18 and its variants 50 Determination of value

[0321] The three peptides with the highest activity identified in the above assays were tested in an L-929 cell killing assay (as described in Example 4 above), with peptide concentrations ranging from 50 μmol to 0.78125 μmol (serial two-fold dilutions), and the EC50 of each peptide was measured. 50 Values ​​(data not shown). The following peptides were tested: DD-18: DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) DD-18-KtoR: DDCVWQP R FNNYWC-amide (SEQ ID NO: 39) Biotin-DD-18-KtoR: Biotin-DDCVWQP R FNNYWC-amide (SEQ ID NO: 39)

[0322] EC of each peptide 50 Values are as follows: DD-18: 16.1 micromolar DD-18KtoR: 6.16 micromolar Biotin-DD-18KtoR: 8.7 micromolar

[0323] Example 11: Peptide phage library Lasso CX 10 Results of sequence analysis of C

[0324] Peptide 18 was isolated from the phage library Lasso CX 10 C (CX2X3X4X5X6X7X8X9X 10 X 11 C). Deep sequence analysis of peptide matches from this library revealed that the most frequently occurring amino acids at each position (from left to right between cysteine residues) of the peptide were as follows:

[0325] Peptides from this library always have the FNN(W / Y) amino acid sequence at positions X7 to X 10 of the peptide.

[0326] Example 12: Comparison of Peptide 18 and Variants for Binding in the TNFα Binding Assay

[0327] Peptide 18 and the following variants (with altered spacing between the biotin molecule and the AspAsp (DD) N-terminal tag) were tested for TNFα binding using the TNFα binding assay described in Example 2. N-terminal spacing variants with PEG4 (4 PEG subunits) were made by inserting PEG4 at the N-terminus between the biotin and the DD core TNFα binding domain of peptide-18. The following peptides were tested: Biotin-DD-18-KtoR: Biotin-DDCVWQP R FNNYWC-amide (SEQ ID NO: 39) Biotin-DD-18: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) Biotin-PEG4-DD-18: Biotin-PEG4-DDCVWQPKFNNYWC-amide (SEQ ID NO: 54)

[0328] See Figure 9The addition of a PEG4 spacer at the N-terminus of peptide 18 did not significantly affect the binding of the peptide to TNFα (compare left-most to third columns from left). In addition, two different preparations of the biotinylated version of the peptide, biotin-DD-18-KtoR, were tested for binding to TNFα. Little difference in TNFα binding was detected (compare left-most column to second column from right).

[0329] Peptide 18 is cyclized by forming a disulfide bond between the terminal cysteine residues of the core TNFα binding domain. To determine whether a single amino acid deletion or insertion between the cysteine residues would alter the activity of the peptide, a valine-deleted peptide (biotin-DD-18-noVal) and an alanine-inserted peptide (biotin-DD-18-plusAla) were prepared. The sequences are as follows: Biotin-DD-18-noVal: Biotin-DDCWQPKFNNYWC-amide (SEQ ID NO: 42) Biotin-DD-18-plusAla: Biotin-DDC A VWQPKFNNYWC-amide (SEQ ID NO: 43)

[0330] Again, see Figure 9 The deletion or insertion of an amino acid residue adjacent to the conserved N-terminal cysteine residue significantly reduced the binding of the peptide to TNFα (compare column 2 to columns 4 and 5). Peptides biotin-DD-18-noVal (SEQ ID NO: 43) and biotin-DD-18-plusAla (SEQ ID NO: 44) were also tested in the L-929 cell killing assay and were shown to be almost incapable of inhibiting TNFα-induced cell killing (data not shown). This result indicates that the spacing of certain residues in the core TNFα binding domain and the conformation of the peptide can be important for binding to TNFα.

[0331] Example 13: Comparison of PEG-modified peptide 18 and a trimeric version of peptide 18 in the L-929 cell killing assay.

[0332] TNFα can associate into trimers in vivo. To determine whether multimerization affects the activity of the peptide, peptide 18 and its variants were tested in the L-929 cell killing assay described in Example 4. First, a monomeric peptide 18 with a Lys to Arg substitution in the core TNFα binding domain (peptide DD-18KtoR; SEQ ID NO: 39) was tested with or without N-terminal Lys-PEG12 (12 PEG subunits) or C-terminal peg12-Lys additions. The monomeric peptide has the following structure: DD-18-KtoR: DD CVWQPRFNNYWC -amide (SEQ ID NO: 39) DD-18-KtoR-PEG12-K: Ac-DD CVWQPRFNNYWC-PEG12-K -amide (SEQ ID NO: 44) K-PEG12-DD-18-KtoR: Ac-K-PEG12-DD CVWQPRFNNYWC -amide (SEQ ID NO: 45)

[0333] Referring to Figure 10 The addition of Lys-PEG12 at the N-terminus or PEG12-Lys at the C-terminus did not significantly alter the blockade of TNFa-induced cell killing (compare columns 1 and 8 and 9).

[0334] Peptide 18 (Biotin-DD-18) and its N-terminal PEG4 version were also tested for activity. The structures of these peptides are as follows: Biotin-DD-18: Biotin-DD CVWQPKFNNYWC -amide (SEQ ID NO: 32) Biotin-PEG4-DD-18: Biotin-PEG4-DD CVWQPKFNNYWC -amide (SEQ ID NO: 54)

[0335] Referring to Figure 10 The addition of PEG4 to the N-terminus of monomeric peptide 18 did not significantly alter the blockade of TNFa-induced cell death (compare columns 2 and 3 from left).

[0336] Peptide 18 (DD-18) was trimerized by a method of assembly on a solid phase resin to form the peptide DD-18-tricasso. Semi-orthogonal protecting groups (Dde and Fmoc) were used to directly assemble the trimeric peptide DD-18 tricasso (see WO2017 / 040350 for a general description of the tricasso trimerization method). In addition, peptide 18 with a Lys to Arg substitution (DD-18-KtoR; SEQ ID NO: 39) was trimerized by a naturally forming trimeric IZ coiled coil sequence (IKKEIEAIKKEQEAIKKKIEAIEKEA (SEQ ID NO: 46)) and a PEG12 spacer to form the peptide DD-18-KtoR-IZ. The IZ forms a non-covalent trimer. The structures of the resulting peptides are as follows: DD-18-tricasso: [DD CVWQPKFNNYWC-PEG12]3-KK -amide DD-18-KtoR-IZ: DDCWQPRFNNYWC-PEG12-IKKEIEAIKKEQEAIKKKIEAIEKEA-hydrazide (SEQ ID NO: 39 linked to SEQ ID NO: 47 by PEG12)

[0337] Referring again to Figure 10 Trimerization of these peptides did not significantly reduce the ability of the peptides to block TNFα-induced cell death (compare columns 2 and 6 and columns 1 and 7).

[0338] The assay also tested a valine deletion (Biotin-DD-18-noVal) and an alanine insertion (Biotin-DD-18-plusAla) of peptide 18. The sequences are as follows: Biotin-DD-18-noVal: Biotin-DDCWQPKFNNYWC-amide (SEQ ID NO: 42) Biotin-DD-18-plusAla: Biotin-DDCAVWQPKFNNYWC-amide (SEQ ID NO: 43)

[0339] Referring again to Figure 10 Deletion or insertion of amino acid residues adjacent to the conserved N-terminal cysteine residue significantly reduced the binding of the peptides to TNFα (compare columns 2, 4, and 5 from the left).

[0340] The EC50values of each of the trimer peptides DD-18-tricasso and DD-18-KtoR-IZ were determined in the L-929 cell killing assay by varying the peptide concentration from 50 micromolar to 0.78125 micromolar (two-fold serial dilutions). 50 The EC50values of the trimer peptide DD-18-KtoR-IZ were determined to be 2.4 micromolar and 2.1 micromolar in PBS and water, respectively. The EC50value of the peptide DD-18-tricasso was determined to be 3.4 micromolar. 50 The EC50values of the trimer peptide DD-18-KtoR-IZ were determined to be 2.4 micromolar and 2.1 micromolar in PBS and water, respectively. The EC50value of the peptide DD-18-tricasso was determined to be 3.4 micromolar. 50 The EC50values of the trimer peptide DD-18-KtoR-IZ were determined to be 2.4 micromolar and 2.1 micromolar in PBS and water, respectively. The EC50value of the peptide DD-18-tricasso was determined to be 3.4 micromolar.

[0341] Example 14: Preparation and testing of covalently linked dimer and trimer peptides

[0342] Covalently linked dimers and trimers of the peptide DD-18-KtoR were prepared and tested for activity in the L-929 cell killing assay. Covalently linked dimers and trimers of DD-18-KtoR were prepared using amine-NHS chemistry. DD-18-KtoR monomers were prepared with terminal amine groups by inserting a lysine residue and a PEG group at either the N- or C-terminus of the peptide. Dimers and trimers were then prepared using NHS-amine chemistry using linkers with multiple NHS groups. The dimers and trimers were purified using standard HPLC purification methods.

[0343] Two different DD-18-KtoR monomers were prepared in this study: N-terminal or C-terminal lysine was added and spaced from the DD-18-KtoR peptide by a PEG12 spacer (SEQ ID NOs: 45 and 46). To prepare dimers, DD-18-KtoR peptides containing PEG12-Lys were reacted with the NHS-PEG6-NHS linker. To prepare trimers, DD-18-KtoR peptides containing PEG12-Lys were reacted with the Fmoc-amino-tri-NHS linker; in this case, the Fmoc was retained on the amine of the trimer linker.

[0344] Peptides tested in the L-929 cell killing assay were as follows: DD-18-KtoR: DDCVWQPRFNNYWC-amide (SEQ ID NO: 39) DD-18-KtoR-N-dimer: (Ac-K-PEG12-DDCVWQPRFNNYWC-amide)2-PEG6 DD-18-KtoR-C-dimer: (Ac-DDCVWQPRFNNYWC-PEG12-K-amide)2-PEG6 DD-18-KtoR-N-trimer: Fmoc-[Ac-K-PEG12-DDCVWQPRFNNYWC-amide]3 DD-18-KtoR-C-trimer: Fmoc-[Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3

[0345] See Figure 11 Both the covalently linked dimers and trimers of the peptide DD-18-KtoR exhibited more significant inhibition of TNFα-mediated cell killing than the monomeric peptide DD-18-KtoR. The activity of the dimers and trimers was also higher than the anti-TNFα VHH2 nanobody control.

[0346] Example 15: EC50 determination of dimers and trimers of the peptide DD-18-KtoR 50 values

[0347] To determine the EC50 of the dimer and trimer of DD-18-KtoR prepared in Example 14 50 In the L-929 cell killing assay, the concentrations of the following dimers and trimers (SEQ ID NO: 45 and 46) of the peptide DD-18-KtoR were varied from 10 μmol to 0.0015 μmol (eight 2-fold dilutions). The peptides tested are as follows: DD-18-KtoR-N-dimer: (Ac-K-PEG12-DDCVWQPRFNNYWC-amide)2-PEG6 DD-18-KtoR-C-dimer: (Ac-DDCVWQPRFNNYWC-PEG12-K-amide)2-PEG6 DD-18-KtoR-N-trimer: Fmoc-[Ac-K-PEG12-DDCVWQPRFNNYWC-amide]3 DD-18-KtoR-C-trimer: Fmoc-[Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3

[0348] Calculated EC 50 The values ​​are as follows: DD-18-KtoR-N-dimer: 10 nM DD-18-KtoR-C-dimer: 2.6 nM DD-18-KtoR-N-trimer: 2nM DD-18-KtoR-C-trimer: << 1.5 nM

[0349] Both N-linked and C-linked peptides are more potent in trimers than in dimers. Conversely, C-linked peptides, whether dimers or trimers, are more potent than N-linked peptides.

[0350] Example 16: Determination of species selectivity of DD-18-KtoR trimer

[0351] The C-terminal linked DD-18-KtoR trimer was tested for binding activity to TNFα proteins from multiple species in an L-929 cell killing assay: Recombinantly expressed soluble TNFα from human, monkey, dog, and rat were tested at concentrations ranging from 10 micromolar to 41 nM (three-fold dilutions). TNFα from these species are capable of binding to human TNFR, so the L-929 cell killing assay can be used to assess the blocking activity of the peptide DD-18-KtoR to different TNFα proteins. The trimer used was DD-18-KtoR-C-trimer: Fmoc-[Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3.

[0352] The C-terminal linked trimer was active against TNFα from human, monkey, and dog at all concentrations tested in the cell killing assay (data not shown). The trimer was not active against rat TNFα at any of the concentrations tested in this assay (data not shown).

[0353] Example 17: Identification of residues in monomeric peptide DD-18 involved in interaction with TNFα

[0354] To identify residues in the peptide DD-18 involved in its interaction with TNFα, additional mutants of the peptide DD-18 (peptide 18) were prepared. These peptides were tested in the TNFα binding assay described in Example 2. The peptides tested are as follows, with differences from the peptide Bio-DD-18 underlined: Biotin-DD-018: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) Biotin-DD-018(K2R): Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 39) R FNNYWC-amide (SEQ ID NO: 39) Bio-018-WtoG: Biotin-DDCV G QPKFNNYWC-amide (SEQ ID NO: 47) Bio-018-NtoG: Biotin-DDCVWQPKFN G YWC-amide (SEQ ID NO: 48)

[0355] In addition, the N-terminal DD tag in the following peptides was changed to DDD: Bio-DDD-018: Biotin- D DDCVWQPKFNNYWC-amide (SEQ ID NO: 49)

[0356] See Figure 12peptides with substitutions of Trp and Asn to Gly (positions 3 and 9, respectively, of the core TNFa binding domain) had significantly reduced binding activity (compare column 11 with columns 1 and 2). As shown previously, peptide Biotin-DD-18K2R (with a substitution of Lys to Arg at position 6 of the core TNFa binding domain; SEQ ID NO: 39) exhibited higher binding activity than peptide Bio-DD-18 (SEQ ID NO: 32) (compare columns 10 and 11). The addition of one extra D-Asp residue at the N-terminal end of peptide Biotin-DD-018 (peptide Bio-DDD-018; SEQ ID NO: 50) reduced its binding activity to some extent (compare columns 3 and 11 from the left).

[0357] In addition, other hits from the CX 10 C peptide library were also tested in the TNFa binding assay. The peptides tested were as follows, with the differences from peptide Bio-DD-18 underlined: Bio-DD-Hit19: Biotin-DDC QF QP R FNN WQ C-amide (SEQ ID NO: 50) Bio-DD-Hit33: Biotin-DDC HFSQR FNN W WC-amide (SEQ ID NO: 51) Bio-DD-Hit92: Biotin-DDC AY Q RQ FNN W WC-amide (SEQ ID NO: 52) Bio-DD-Hit193: Biotin-DDC WFEHR FNN WH C-amide (SEQ ID NO: 53)

[0358] Again, referring to Figure 12 In comparison to peptide DD-018, peptides Bio-DD-Hit19, Bio-DD-Hit92 and Bio-DD-Hit193 exhibited some, but reduced, TNFa binding activity, while peptide Bio-DD-Hit33 exhibited significant binding activity (compare columns 6, 7 and 9 with column 11 from the left).

[0359] To assess the contribution of proximity of the N-terminal DD tag to the core TNFa binding domain, a PEG4 molecule was inserted between the DD tag and the N-terminus of the core TNFa binding domain. The activity of this peptide was compared to a peptide in which a PEG4 molecule was added to the N-terminus of the DD tag. The peptides tested were as follows: Bio-DD-PEG4-018: Biotin-DD-PEG4-CVWQPKFNNYWC-amide (SEQ ID NO: 55) Bio-PEG4-DD-018: Biotin-PEG4-DDCVWQPKFNNYWC-amide (SEQ ID NO: 54)

[0360] Referring again to Figure 12 , separation of the DD tag from the N-terminus of the core TNFa peptide binding domain reduced TNFa binding activity to some extent (compare 4th, 5th, and 11th bars from left).

[0361] Example 18: Modification of the N-terminal DD tag of monomeric peptide Bio-DD-18

[0362] To further assess the impact of sequence and proximity changes of the N-terminal DD tag to the core TNFa binding domain, the following peptides were tested in the L-929 cell killing assay and the EC 50 values were determined: Biotin-PEG4-DD-018: Biotin-PEG4-DDCVWQPKFNNYWC-amide (SEQ ID NO: 54) Biotin-DD-018(K2R): Biotin-DDCVWQPRFNNYWC-amide (SEQ ID NO: 39) Bio-DDD-018: Biotin-DDDCVWQPKFNNYWC-amide (SEQ ID NO: 49) Bio-DD-018: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) Bio-D-018: Biotin-DCVWQPKFNNYWC-amide (SEQ ID NO: 56) Bio-DD-PEG4-018: Biotin-DD-PEG4-CVWQPKFNNYWC-amide (SEQ ID NO: 55)

[0363] The L-929 cell killing assay was performed as described in Example 4.

[0364] EC 50 values were as follows: Bio-DD-018: 16.6 micromolar Bio-DDD-018: 4.1 micromolar Bio-D-018: 38 micromolar Biotin-DD-018 (K2R): 2.1 micromolar Biotin-PEG4-DD-018: 15 micromolar Bio-DD-PEG4-018: >50 micromolar

[0365] Referring to these results, the addition of an extra Asp residue in the N-terminal tag (SEQ ID NO: 50) resulted in a more potent (lower) EC 50 value compared to the peptide Bio-DD-018 (SEQ ID NO: 32), while removal of the Asp residue (SEQ ID NO: 57) resulted in a less potent (higher) EC 50 value. Increasing the spacing between the DD tag and the core TNFa binding domain resulted in a less potent (higher) EC 50 value. Finally, as shown previously, the EC 50 value of the peptide Biotin-DD-018 (K2R; SEQ ID NO: 39) was lower than that of the peptide Bio-DD-018.

[0366] Example 19: Thermal stability of TNFa complexes with monomeric peptide Biotin-DD-18 and selected variants

[0367] The thermal stability of TNFa complexes with the peptide Biotin-DD-18 and the following mutants was determined using the high-throughput thermal scan method described in Lavinder et al., J. Am. Chem. Soc. 2009, 131(11): 3794-379. Briefly, the fluorescent dye SYPRO TM binds to protein (or protein-peptide) complexes in solution. Upon denaturation of the complex, the dye fluoresces. Results are expressed as TNFa-ATm (degrees Celsius).

[0368] For this study, the following peptides were complexed with TNFa, with the differences from Biotin-DD-18 underlined: Bio-18: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) Bio-18-WtoG-1: Biotin-DDCVWQPKFNNY G C-amide (SEQ ID NO: 35) Bio-18-WtoG-2: Biotin-DDCV GQPKFNNYWC-amide (SEQ ID NO: 47) Bio-18-NtoG: Biotin-DDCVWQPKFN G YWC-amide (SEQ ID NO: 48)

[0369] The results were as follows: Bio-18: 80.5 (5.7 increase over TNFα alone) Bio-18-WtoG-1 : 75.1 (0.3 increase over TNFα alone) Bio-18-WtoG-2: 75.0 (0.2 increase over TNFα alone) Bio-18-NtoG: 74.7 (0.1 decrease over TNFα alone) TNFα alone: 74.8

[0370] Each single amino acid substitution in Biotin-DD-18 decreased the thermal stability of the TNFα / peptide complex, consistent with the decreased binding activity of these peptides, as shown previously.

[0371] Example 20: Thermal stability of monomeric peptide Biotin-DD-18 with spacer variations

[0372] The thermal stability of DD spacer variations on the peptide Biotin-DD-18 was determined using the thermal stability assay described in Example 19. The peptides tested were as follows: Biotin-PEG4-DD-018: Biotin-PEG4-DDCVWQPKFNNYWC-amide (SEQ ID NO: 54) Bio-DD-PEG4-018: Biotin-DD-PEG4-CVWQPKFNNYWC-amide (SEQ ID NO: 55)

[0373] The results were as follows: Biotin-PEG4-DD-018: 80.3 (5.5 increase over TNFα alone) Bio-DD-PEG4-018: 78.4 (3.6 increase over TNFα alone) TNFα alone: 74.8

[0374] These results are consistent with the analysis of these peptides above, indicating that the introduction of a PEG4 spacer between the DD tag and the N-terminus of the core TNFα binding domain attenuates TNFα binding and complex stability.

[0375] Example 21: Thermal stability of monomeric peptides with N-terminal DD tag modifications of peptide DD-18

[0376] The thermal stability assay described in Example 19 was performed on the following peptides to further evaluate sequence changes in the N-terminal DD tag of peptide Biotin-DD-18. The following peptides were tested: DD-018: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) D-018: Biotin-DCVWQPKFNNYWC-amide (SEQ ID NO: 56) DDD-018: Biotin-DDDCVWQPKFNNYWC-amide (SEQ ID NO: 49)

[0377] The results were as follows: DD-018: 80.5 (5.7 higher than TNFa alone) D-018: 78.9 (4.1 higher than TNFa alone) DDD-018: 81.6 (6.8 higher than TNFa alone) TNF: 74.8

[0378] These results are consistent with the analysis of these peptides described above, indicating that increasing the number of Asp residues in the N-terminal tag increases TNFa binding and complex stability.

[0379] Example 22: Thermal stability of peptide Bio-DD-018 and variants

[0380] The thermal stability assay described in Example 19 was performed on the following peptides from the library screen described in Example 1 to further evaluate the effect of sequence changes in peptide Biotin-DD-18 on TNFa binding. The following peptides were tested, with differences from peptide Biotin-DD-18 underlined: DD-018: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) DD-018-KtoR: Biotin-DDCVWQP R FNNYWC-amide (SEQ ID NO: 39) DD-Hit19: Biotin-DDC QF QP R FNN WQ C-amide (SEQ ID NO: 50) DD-Hit33: Biotin-DDC HFSQR FNN W WC-amide (SEQ ID NO: 51) DD-Hit92: Biotin-DDC AY QRQ FNN W WC-amide (SEQ ID NO: 52) DD-Hit193: Biotin-DDC WFEHR FNN WH C-amide (SEQ ID NO: 53) DD-Hit16: Biotin-DDC HFN P R FNN W WC-amide (SEQ ID NO: 41)

[0381] The results were as follows (in °C): DD-018: 80.5 (5.7 higher than TNFa alone) DD-018-KtoR: 81.9 (8.1 higher than TNFa alone) DD-Hit19: 76.6 (1.8 higher than TNFa alone) DD-Hit33: 77.5 (2.7 higher than TNFa alone) DD-Hit92: 77.2 (2.7 higher than TNFa alone) DD-Hit193: 75.9 (1.1 higher than TNFa alone) DD-Hit16: 79.9 (5.1 higher than TNFa alone) TNFa alone: 74.8

[0382] Although the thermal stability of peptide DD-Hit16 was close to that of peptide DD-018, the peptides representing other matches in the library exhibited lower thermal stability than peptides Bio-DD-18 and Bio-DD-018-KtoR.

[0383] Example 23: Analysis of the effect of varying the length of the PEG spacer in the peptide DD-18-KtoR trimer

[0384] Trimer with different PEG spacers were prepared to determine the effect of PEG spacer length on the activity of the peptide DD-18-KtoR (SEQ ID NO: 39) trimer in the L-929 cell killing assay. The peptide DD-018-KtoR-C-trimer (PEG12) from Example 14 was used. Two different preparations of the 018-KtoR-C-trimer (PEG12) were tested in this study. They were designated -1 and -2, respectively. In addition, trimer with PEG spacers of 4 and 8 PEG subunits were prepared. The trimer peptides tested were as follows: 018-KtoR-C-trimer (PEG12) - 1 : Fmoc-[Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3 018-KtoR-C-trimer (PEG12) - 2: Fmoc-[Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3 018-KtoR-C-trimer (PEG4): Fmoc-[Ac-DDCVWQPRFNNYWC-PEG4-K-amide]3 018-KtoR-C-trimer (PEG8): Fmoc-[Ac-DDCVWQPRFNNYWC-PEG8-K-amide]3

[0385] See Figure 13 The activity of the PEG8 spacer and the PEG12 spacer was approximately the same, while the binding activity of the PEG4 spacer was consistently lower (compare bars 2, 3, and 4 to bar 1 in each series).

[0386] Example 24: Activity of Peptides DD-18-Dimer and Trimer in Cell Killing Assay Using Mouse TNF

[0387] In Example 16, Peptide 018-KtoR-C-trimer and Peptide 018-KtoR-C-dimer showed binding to human, monkey, and canine TNFa, but not to rat TNFa. In this study, the dimer and trimer were tested against mouse TNFa in an L-929 cell killing assay.

[0388] The peptides tested were as follows: DD-18-KtoR-N-dimer: (Ac-K-PEG12-DDCVWQPRFNNYWC-amide)2-PEG6 DD-18-KtoR-C-dimer: (Ac-DDCVWQPRFNNYWC-PEG12-K-amide)2-PEG6 DD-18-KtoR-N-trimer: Fmoc-[Ac-K-PEG12-DDCVWQPRFNNYWC-amide]3 DD-18-KtoR-C-trimer: Fmoc-[Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3

[0389] In this study, neither the dimer nor the trimer exhibited significant binding activity to mouse TNFa (data not shown).

[0390] Example 25: Modifications of Peptide DD-18 Sequence

[0391] from CX 10 Peptide 001 was a strong binder in phage display screening, but was insoluble under the aqueous assay conditions used in these studies. Based on the similarity of peptide 001 to peptide 018-KtoR, a modified peptide 018 was designed. The core TNFα binding domain sequences of peptide 001, peptide 018-KtoR, and modified peptide 18 are shown below, with the differences from peptide 018-KtoR underlined: Peptide 001: C TF Q W RFNNYWC (SEQ ID NO: 12) Peptide 018-KtoR: CVWQPRFNNYWC (SEQ ID NO: 5) Modified peptide 18-KtoR: C TF QPRFNNYWC (SEQ ID NO: 6)

[0392] Another variant of peptide DD-018 was also tested; this variant has an N to Q substitution near the C-terminal end of the peptide (at the penultimate C-terminal position of the core TNFα binding domain). These peptides were tested in the TNFα binding assay (Example 2). Because of the sequence similarity of peptide DD-018 to peptide 001 from CX 10 Another peptide Hit 14 from the C library, which has sequence similarity to peptide DD-018, was also tested. The peptides used in this study are shown below, with the differences from peptide biotin-DD-018 underlined: Biotin-DD-018: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) Biotin-DD-018-KtoR: Biotin-DDCVWQP R FNNYWC-amide (SEQ ID NO: 39) Biotin-DD-018-NtoQ: Biotin-DDCVWQPKFN Q YWC-amide (SEQ ID NO: 57) Biotin-DD-TF-018-KtoR: Biotin-DDC TF QP R FNNYWC-amide (SEQ ID NO: 58) Biotin-DD-Hit 14: Biotin-DDCV F Q HH FNN W WC-amide (SEQ ID NO: 59)

[0393] Two different human TNFα preparations were used in this study to test each peptide.

[0394] See Figure 14 , the assay results for each TNFα preparation are shown for each test peptide (left bar and right bar within each pair). Peptide Biotin-DD-018-KtoR exhibited slightly better TNFα binding than peptide Biotin-DD-018, which is consistent with previous results. Peptide Biotin-DD-018-NtoQ exhibited little TNFα binding activity compared to peptide Biotin-018-KtoR. This result indicates that residues at the C-terminal end of the peptide are involved in contact with TNFα. The modified peptide, Biotin-DD-TF-018-KtoR, exhibited slightly better binding to TNFα than peptide Biotin-DD-018-KtoR. Hit 14 exhibited slightly lower binding than peptide Biotin-DD-018.

[0395] Example 26: Determination of EC 50 values for modified DD-018-KtoR peptides using L-929 cell killing assay

[0396] To further analyze the effect of modifications to peptide Biotin-DD-018, the following peptides were tested in the L-929 cell killing assay to determine EC 50 values, where the differences from peptide Biotin-DD-018 are underlined: Bio-DD-018: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) Bio-DDD-018-KtoR: Biotin- D DDCVWQP R FNNYWC-amide (SEQ ID NO: 60) Bio-DD-018-KtoR: Biotin-DDCVWQP R FNNYWC-amide (SEQ ID NO: 39) Bio-DD-TF-018-KtoR: Biotin-DDC TF QP R FNNYWC-amide (SEQ ID NO: 58) Bio-DD-Hit14: Biotin-DDC VF Q HH FNN W WC-amide (SEQ ID NO: 59)

[0397] EC 50 values were as follows: Bio-DD-018: 14.5 micromolar Bio-DDD-018-KtoR: 2.7 micromolar Bio-DD-018-KtoR: 5.4 micromolar Bio-DD-TF-018-KtoR: 1.7 micromolar Bio-DD-hit14: 14.7 micromoles

[0398] These results indicate that TF substitution near the N-terminal cysteine ​​residue in the peptide Bio-DD-TF-018-KtoR enhances its activity. Furthermore, the addition of an additional aspartic acid residue to the N-terminal tag improves (decreases) the peptide's EC50. 50 Value. Hit14 exhibits EC values ​​similar to peptide biotin-DD-018. 50 value.

[0399] Example 27: Analysis of further modifications to the peptide Bio-DD-018-KtoR

[0400] Based on the results of the previous embodiment, other peptides containing TF substitutions or variants thereof were designed. These peptides were tested in an L-929 cell killing assay to determine their EC50. 50 Values. The peptides tested are shown below, with differences from peptide Bio-DD-018-KtoR underlined: Bio-DD-018-KtoR: Biotin-DDCVWQPRFNNYWC-amide (SEQ ID NO: 39) Bio-DD-TF-018-KtoR: Biotin-DDC TF QPRFNNYWC-amide (SEQ ID NO: 58) Bio-DD-TF-018-KtoR-W: biotin-DDC TF QPRFNN W WC-amide (SEQ ID NO: 61) Bio-DD-VF-018-KtoR: Biotin-DDCV F QPRFNNYWC-amide (SEQ ID NO: 62) Bio-DD-SF-018-KtoR: Biotin-DDC SF QPRFNNYWC-amide (SEQ ID NO: 63)

[0401] EC 50 The value is determined as follows: Bio-DD-018-KtoR: 2.25 micromolar Bio-DD-TF-018-KtoR: 1.16 micromoles Bio-DD-TF-018-KtoR-W: 0.54 micromolar Bio-DD-VF-018-KtoR: 0.64 micromolar Bio-DD-SF-018-KtoR: 2.78 micromolar

[0402] These results also show that both TF substitution and F substitution in the N-terminus of the core TNFa binding domain increase its activity. Both Tyr and Trp in the second C-terminal position of the core TNFa binding domain allow comparable binding activity to be achieved.

[0403] Example 28: Comparison of the activity of the peptide DD-TF-018-KtoR trimer with anti-TNFa antibodies and VHH2 in the L-929 cell killing assay

[0404] The activity of a trimeric form of DD-TF-018-KtoR (SEQ ID NO: 59) with a PEG8-K spacer attached to each arm was compared to other TNFa blockers in the L-929 cell killing assay to determine the EC 50 values. Anti-TNFa antibodies were from R&D Systems (research grade). Anti-TNFa VHH2 nanobody is described in Beirnaert et al., 2017, Front. Immunol. 8:867. The peptides tested were as follows: DD-TF-018-KtoR C-trimer (PEG8): Fmoc-[Ac-DDCTFQPRFNNYWC-PEG8-K-amide]3

[0405] The EC 50 values were as follows: DD-TF-018-KtoR C-trimer (PEG8): 1 pM to 1 fM Anti-TNFa antibody: 90.5 pM Anti-TNFa VHH2 nanobody: 16.9 pM

[0406] In this assay, the peptide DD-TF-018-KtoR trimer was more effective than both the anti-TNFa antibody and the anti-TNFa VHH2.

[0407] Example 29: Analysis of changes in trimer backbone structure and spacer length in the L-929 cell killing assay

[0408] To determine whether different trimeric scaffolds affect and / or how they affect the activity of the peptide DD-018-KtoR (SEQ ID NO: 32), three different scaffolds were tested. In addition, the peptide DD-TF-018-KtoR (SEQ ID NO: 58) was included in one trimeric construct. See Figure 15A The scaffolds tested were Fmoc-scaffold (Quanta BioDesign), Fmoc-PEG27scaffold (Quanta BioDesign), and cyclohex (cyclohexane tricarboxylic acid (also known as 1,3,5-cyclohexane tricarboxylic acid) was prepared by converting the tricarboxylic acid compound with NHS (N-hydroxysuccinimide) in the presence of a carbodiimide (e.g., DIC or DCC)). The Fmoc-scaffold has been used previously in Example 14. In addition, the length of the PEG spacer was changed from PEG8 to PEG6. The trimeric peptides were tested in the L-929 cell killing assay using a peptide concentration of 0.2 micromolar to 0.51 pM (5-fold dilutions). The trimeric peptides tested were as follows: 018-KtoR-C-trimer (PEG6)-Fmoc: [Ac-DDCVWQPRFNNYWC-PEG6-K-amide]3-Fmoc 018-KtoR-C-trimer (PEG12)-PEG27-Fmoc: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-PEG27-Fmoc 018-KtoR-C-trimer (PEG12)-Fmoc: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-Fmoc DD-TF-018-KtoR-trimer (PEG12)-Fmoc: [Ac-DDCTFQPRFNNYWC-PEG12-K-amide]3-Fmoc 018-KtoR-trimer (PEG12)-cyclohex: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-cyclohex

[0409] See Figure 15B The choice of scaffold did not appear to significantly affect the activity of the trimer, although there was a decrease in activity for the trimer with the PEG6 spacer compared to the PEG8 spacer tested previously (see Example 23).

[0410] Example 30: Further analysis of changes in trimeric backbone structure in L-929 cell killing assay

[0411] To determine if other trimeric backbones increased the activity of the peptide DD-018-KtoR (SEQ ID NO: 39) in the trimeric form, five different backbones were tested in addition to the Fmoc-PEG27 backbone used in Example 29. See Figure 16A The backbones tested were nitro-backbone (prepared from 4-(2-carboxyethyl)-4-nitro-pimelic acid by converting the tricarboxylic acid compound with NHS (N-hydroxysuccinimide) in the presence of a carbodiimide (e.g., DIC or DCC)), amine-PEG27-backbone (Quanta BioDesign), Fmoc-backbone (see Example 29), amine-backbone (Quanta BioDesign), cholestrol-backbone (Quanta BioDesign), and biotin-PEG27-backbone (Quanta BioDesign). The trimeric peptides were tested in the L-929 cell killing assay using a peptide concentration of 0.2 micromolar to 2.6 pM (5-fold dilutions). The trimeric peptides tested were as follows: 018-KtoR-C-trimer (PEG12)-nitro: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-nitro 018-KtoR-C-trimer (PEG12)-PEG27-amine: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-PEG27-amine 018-KtoR-C-trimer (PEG12)-Fmoc: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-Fmoc 018-KtoR-C-trimer (PEG12)-amine: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-amine 018-KtoR-C-trimer (PEG12)-PEG27-biotin: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-PEG27-biotin 018-KtoR-C-trimer (PEG12)-PEG27-cholesterol: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-PEG27-cholesterol

[0412] See Figure 16B Generally, the choice of trimer backbone did not significantly affect the activity of the trimer peptides, although the cholesterol-backbone produced slightly lower activity at lower concentrations.

[0413] Example 31: Activity of certain trimer peptides at lower concentrations

[0414] Using the Fmoc-, amine-, or cyclohex- backbones from Examples 29 and 30, the trimer of peptide DD-018-KtoR (SEQ ID NO: 39) or peptide TF-DD-018-KtoR (SEQ ID NO: 59) were tested at lower concentrations than in the previous examples in the L-929 cell killing assay to determine the EC 50 values. The trimer peptides were tested at concentrations ranging from 0.32 nM to 0.16 fM (5-fold dilutions). The trimer peptides tested were as follows: 018-KtoR-C-trimer (PEG12)-Fmoc: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-Fmoc 018-KtoR-C-trimer (PEG12)-amine: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-amine 018-KtoR-C-trimer (PEG12)-cyclohex: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-cyclohex TF-018-KtoR-C-trimer (PEG12)-Fmoc: [Ac-DDCTFQPRFNNYWC-PEG12-K-amide]3-Fmoc

[0415] EC 50 values were as follows: 018-KtoR-C-trimer (PEG12)-Fmoc: 5.1 pM 018-KtoR-C-trimer (PEG12)-amine: 0.16 fM 018-KtoR-C-trimer (PEG12)-cyclohex: 4 pM TF-018-KtoR-C-trimer (PEG12)-Fmoc: 0.16 fM

[0416] 018-KtoR-C-trimer (PEG12)-amine and TF-018-KtoR-C-trimer (PEG12)-Fmoc produced comparable activity. In the Fmoc-trimer form, TF-018-KtoR trimer produced superior activity to the 018-KtoR trimer. For the 018-KtoR trimer, the amine trimer produced the best activity.

[0417] Example 32: Analysis of further modifications to monomeric DD-VF-018-KtoR-WW peptide in the L-929 cell killing assay

[0418] Based on the above examples, the peptide TF-018-KtoR-WW (DDCTFQPRFNNWWC; SEQ ID NO: 61) produced the best activity in the L-929 cell killing assay. Using a similar reference sequence, the peptide VF-018-KtoR-WW (DDCVFQPRFNNWWC; SEQ ID NO: 64) was subjected to additional substitutions and tested in the L-929 cell killing assay. The peptides tested are shown below, with the differences from the peptide VF-018-KtoR-WW (SEQ ID NO: 65) underlined. The peptide Bio-018-KtoR (SEQ ID NO: 39) was also included as a control. Bio-VF-018-KtoR-WW: Biotin-DCVFQPRFNNWWC-amide (SEQ ID NO: 64) Bio-DtoE-VF-018-KtoR-WW: Biotin- E DCVFQPRFNNWWC-amide (SEQ ID NO: 65) Bio-VF-018-QtoK-KtoR-WW: Biotin-DDCVF K PRFNNWWC-amide (SEQ ID NO: 66) VF-018-QtoKbio-KtoR-WW: Ac-DDCVF X 6 PRFNNWWC-amide; wherein X 6 is K(bio); SEQ ID NO: 67) Bio-018-KtoR: Biotin-DDCVWQPRFNNYWC-amide (SEQ ID NO: 39)

[0419] See Figure 17Changing the N-terminal aspartate to glutamate in the tag did not significantly alter the activity of the peptide Bio-VF-018-KtoR-WW (SEQ ID NO: 65). The activity of the peptides VF-018-QtoKbio-KtoR-WW and BioVF-018-QtoKbio-KtoR-WW (SEQ ID NO: 67) (with substitution of Gin (at position 4 of the core TNFa binding domain (counting from the N-terminus)) to Lys or Lys-biotin) was slightly reduced compared to Bio-VF-018-KtoR-WW (SEQ ID NO: 65).

[0420] Example 33: Analysis of further modifications to monomeric DD-VF-018-KtoR-WW peptides in the L-929 cell killing assay

[0421] To determine whether further modifications to the peptide VF-018-KtoR-WW (SEQ ID NO: 65) could improve its activity, additional substitutions were made and tested in the L-929 cell killing assay. The peptides tested are shown below, with differences from the peptide VF-018-KtoR-WW (SEQ ID NO: 65) underlined. Peptide Bio-018-KtoR (SEQ ID NO: 39) was also included as a control. Bio-VF-018-KtoR-WW: Biotin-DDCVFQPRFNNWWC-amide (SEQ ID NO: 64) Bio-VF-018-PtoG-KtoR-WW: Biotin-DDCVFQGRFNNWWC-amide (SEQ ID NO: 68) Bio-VF-018-PtoH-KtoR-WW: Biotin-DDCVFQHRFNNWWC-amide (SEQ ID NO: 69) Bio-VF-018-QtoN-KtoR-WW: Biotin-DDCVFNPRFNNWWC-amide (SEQ ID NO: 70) Bio-018-KtoR: Biotin-DDCVWQPRFNNYWC-amide (SEQ ID NO: 39)

[0422] See Figure 18, peptides Bio-VF-018-PtoG-KtoR-WW, Bio-018-KtoR-WW and Bio-VF-018-PtoH-KtoR-WW had comparable activity at the highest concentration. As the concentration was reduced, peptides Bio-VF-018-KtoR-WW and Bio-VF-018-PtoH-KtoR-WW retained better activity than the other peptides, although all peptides retained comparable binding activity to peptide Bio-018-KtoR.

[0423] Example 34: Analysis of further modifications of peptide DD-018-KtoR in monomeric and dimeric forms in L-929 cell killing assay

[0424] The DD tag at the N-terminus of the peptide is negatively charged at neutral pH, which helps to improve the solubility of the peptide. The two aspartic acid (D) residues in the N-terminal tag maintain high solubility and good activity. In this study, the N-terminal Asp (D) was replaced with a mimetic suc (succinic acid) group. Succinic acid is a dicarboxylic acid. One acid group is conjugated to Asp through an amide bond, while the other acid is free to introduce a negatively charged carboxylate. Monomeric and dimeric forms of peptide DD-018-KtoR-PEG8-K were tested in L-929 cell killing assay. The dimeric form was prepared using amine-NHS chemistry and a NHS-PEG8-NHS crosslinker (QuantaBioDesign). Peptide Bio-DD-TF-018-KtoR-WW (SEQ ID NO: 62) was also included. The peptides tested were as follows: DD-018-KtoR-PEG8-K: Ac-DDCVWQPRFNNYWC-PEG4-PEG4-K-amide (SEQ ID NO: 39) sucD-018-KtoR-PEG8-K: suc-DCVWQPRFNNYWC-PEG4-PEG4-K-amide (SEQ ID NO: 71) DD-018-KtoR-PEG8-K dimer: (Ac-DDCVWQPRFNNYWC-PEG4-PEG4-K-amide)2-PEG6 sucD-018-KtoR-PEG8-K dimer: (suc-DCVWQPRFNNYWC-PEG4-PEG4-K-amide)2-PEG6 Bio-DD-TF-018-KtoR-WW: Biotin-DDCTFQPRFNNWWC-amide (SEQ ID NO: 61)

[0425] See Figure 19 Replacing one Asp residue of the tag with a succinic acid group did not significantly affect the activity of monomer and dimer compared to the unmodified peptide.

[0426] Example 35. Analysis of further modifications to peptide TF-018-KtoR-WW in the L-929 cell killing assay

[0427] In this study, D-a-amino acid analogs were introduced site-selectively to determine if the potency of monomeric peptide TF-018-KtoR-WW (SEQ ID NO: 62) was enhanced in the L-929 cell killing assay. The D-a-amino acid analogs used were penicillamine (Pen), a methionine analog; homoLeu, an analog of leucine (Leu); and norLeu, an analog of leucine (Leu). The peptides tested are as follows, with the differences from peptide Bio-DD-TF-018-KtoR-WW (SEQ ID NO: 62) underlined: Bio-Pen-TF-018-KtoR-WW: Biotin-DD X TFQPRFNNWWC-amide (SEQ ID NO: 72), where X in the peptide is D-penicillamine Bio-TF-018-KtoR-WW-Pen: Biotin-DDCTFQPRFNNWW X - amide (SEQ ID NO: 73), where X in the peptide is D-penicillamine Bio-homoLeu-F-018-KtoR-WW: Biotin-DDC X FQPRFNNWWC-amide (SEQ ID NO: 74), where X in the peptide is D-homoLeucine Bio-norLeu-F-018-KtoR-WW: Biotin-DDC X FQPRFNNWWC-amide (SEQ ID NO: 75), where X in the peptide is D-norLeucine Bio-DD-TF-018-KtoR-WW: Biotin-DDCTFQPRFNNWWC-amide (SEQ ID NO: 61)

[0428] See Figure 20Substitution with D-a-amino acid analogues did not significantly affect the potency of the monomer at the highest concentration, although the peptide Bio-homoLeu-F-018-KtoR-WW exhibited slightly lower activity compared to other peptides.

[0429] Example 36. Thermal stability of TNFa complexes with peptide DD-018-KtoR (SEQ ID NO: 39) and oligomeric variants

[0430] The thermal stability assay described in Example 19 was performed on the following TNFa complexes with peptides to further assess the effect of oligomerization on TNFa binding stability. The following peptides were tested: DD-18-KtoR: DDCVWQPRFNNYWC-amide (SEQ ID NO: 39) DD-18-KtoR-C-dimer: (Ac-DDCVWQPRFNNYWC-PEG12-K-amide)2-PEG6 DD-18-KtoR-C-trimer: Fmoc-[Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3

[0431] In addition, TNFa alone was included as a reference.

[0432] Results were as follows (in °C): DD-18-KtoR: 81.9 (+8.1) DD-18-KtoR-C-dimer 84.6 (+9.8) DD-18-KtoR-C-trimer 86.0 (+10.2) TNFa alone: 74.8

[0433] These results indicate that increasing oligomerization of the peptide DD-018-KtoR improves the thermal stability of the complex of the peptide with TNFa.

[0434] Example 37. Fluorescently labeled peptide DD-018 was used to confirm binding of monomeric peptide to native TNFa (trimer)

[0435] Native TNFa (in trimeric form) was incubated with either peptide DD-018 (SEQ ID NO: 32) or peptide DD-018-WtoG2 (SEQ ID NO: 77) (non-binding control). Both peptides were labeled with an N-terminal fluorescein group (via amide coupling of carboxyfluorescein during solid phase peptide synthesis, SPPS). The mixtures of peptide plus TNFa were then assayed by analytical SEC using an AKTA analytical SEC column in 0.1 M ammonium acetate (pH 8) solution. The peptides tested were as follows: DD-018: Fluor-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) DD-018-WtoG2: Fluor-DDCVGQPKFNNYGC-amide (SEQ ID NO: 76)

[0436] See Figure 21 For each graph, the left Y-axis represents absorbance at 280 nm and the right Y-axis represents absorbance at 480 nm. PI represents the elution peak corresponding to TNFa. P2 represents the elution peak of free peptide. The top graph shows the elution profile of TNFa alone. The middle graph shows the elution profile of TNFa plus the fluor-labeled peptide DD-018. Some of the peptide co-eluted with TNFa (PI), indicating that it bound to TNFa. P2 is the excess peptide. The bottom graph shows the elution profile of TNFa with the fluor-labeled peptide DD-018-WtoG2 (non-binding control). The peptide DD-018-WtoG2 did not co-elute with TNFa.

[0437] Example 38. Peptide TF-018-KtoR-WW and oligomers thereof were tested in the TNFa / TNFR blocking assay and the L-929 cell killing assay.

[0438] The activity of the peptide TF-018-KtoR-WW (SEQ ID NO: 62) as a monomer, dimer, and trimer was determined in the TNFa / TNFR blocking assay (Example 3) and the L-929 cell killing assay (Example 4). The peptides tested were as follows: TF-018-KtoR-WW monomer: Ac-DDCTFQPRFNNWWC-amide (SEQ ID NO: 61) TF-18-KtoR-WW C-dimer: (Ac-DDCTFQPRFNNWWC-PEG12-K-amide)2-PEG6 TF-18-KtoR-WW C-trimer: Amine-[Ac-DDCTFQPRFNNWWC-PEG8-K-amide]3

[0439] In addition, the activity of the peptide monomer and trimer were compared to an anti-TNFa antibody (R&D Systems (Research Grade)).

[0440] Figure 22A A comparison of the binding affinity of the monomer, dimer, and trimer peptides TF-018-KtoR-WW in the TNFa / TNFR blocking assay is shown. The multimerization of the peptide improved the IC 50 . Figure 22BComparison of monomeric trimeric peptide TF-018-KtoR-WW with anti-TNFa antibody (R&D Systems (research grade)) is shown. In the TNFa / TNFR blocking assay, the IC 50 value of the trimeric peptide TF-018-KtoR-WW was superior to the EC

[0441] Figure 23A Comparison of monomeric, dimeric and trimeric peptide TF-018-KtoR-WW in the L-929 cell killing assay is shown. Multimerization of the peptide improved the EC 50 . Figure 23B Comparison of monomeric and trimeric peptide TF-018-KtoR-WW with anti-TNFa antibody (R&D Systems (research grade)) is shown. In this assay, the EC 50 value of the trimeric peptide TF-018-KtoR-WW was superior to the EC 50 value of the anti-TNFa antibody.

[0442] Example 39. Binding affinity determination of peptide TF-018-KtoR-WW monomer and trimer

[0443] Binding affinity of peptide TF-018-KtoR-WW (SEQ ID NO: 62) monomer and trimer was determined using surface plasmon resonance (SPR). Recombinant biotin-TNFa was immobilized on the SPR chip surface by interaction with neutravidin modified chip. Then different concentrations of monomer or trimer were introduced through the chip surface and relative binding parameters were determined. The affinity of the peptide TF-018-KtoR-WW monomer was ~50 nM as determined using monomer concentrations of 1200 nM, 625 nM and 312.5 nM. Due to the stronger interaction of the peptide TF-18-KtoR-WW C-trimer with immobilized TNFa, its affinity could not be determined in this assay (concentrations of 3.75 nM and 1.88 nM were used).

[0444] Example 40. Activity of peptide TF-18-KtoR-WW C-trimer to block membrane bound TNFa

[0445] The ability of the peptide TF-18-KtoR-WW C-trimer to specifically block membrane-bound TNFa activity was tested using an L-929 cell killing assay. HEK293T cells expressing either wild type (WT) or membrane-restricted (Δ1-12aa) TNFa (see Ruuls et al. Immunity 15:533-543, 2001; Decoster et al. J. Biol. Chem. 270:18473-18478, 1995). Membrane-restricted TNFa lacks the substrate cleavage site for TACE (TNFa converting enzyme) (Δ1-12aa). To assess the blocking activity of the TF-18-KtoR-WW C-trimer on membrane-associated TNFa, L-929 (TNFR-expressing) cells were overlaid with HEK293T cell lines expressing either wild type or membrane-restricted (Δ1-12aa) TNFa in the absence or presence of the trimer.

[0446] See Figure 24 In the first panel (far left), the addition of cells expressing WT TNFa resulted in L-929 cell death. In the second panel (left), the addition of cells expressing membrane-restricted TNFa (Δ1-12aa TNFa) resulted in L-929 cell death. In the third panel (left), the addition of cells expressing WT TNFa and the TF-18-KtoR-WW C-trimer rescued L-929 cell death, depending on the amount of TNFa-containing cells added. In the fourth panel (far right), the addition of cells expressing TNFa (Δ1-12aa TNFa) and the TF-18-KtoR-WW C-trimer rescued L-929 cell death.

[0447] In summary, the TF-18-KtoR-WW C-trimer was able to rescue TNFa-mediated cell killing for both WT and membrane-restricted TNFa.

[0448] Example 41. Stability of the peptide TF-18-KtoR-WW C-trimer in simulated gastric and intestinal fluids

[0449] The stability of the peptide TF-18-KtoR-WW C-trimer was evaluated in simulated gastric and intestinal fluids (SGF and SIF, respectively). TF-18-KtoR-WW C-trimer has three PEG8 arms and a free amino group. Simulated gastric fluid (SGF - RICCA Chemicals, product number 7108) contains 0.2% (w / v) sodium chloride in 0.7% (v / v) hydrochloric acid, with 3.2 mg of freshly added purified pepsin (from porcine gastric mucosa, with an activity of 800 to 2500 units per mg of protein) added per milliliter of solution. The pH of the simulated gastric fluid is between 1 and 1.6. Simulated intestinal fluid (SIF - RICCA Chemicals, product number 7109) contains 0.68% (w / v) potassium phosphate, 0.06% (w / v) sodium hydroxide and 1% (w / v) pancreatin. The pH of this solution is between 6.7 and 6.9.

[0450] The 26 mM peptide TF-18-KtoR-WW C-trimer was incubated in SGF and SIF at 37°C for 24 hours, then the amount of peptide TF-18-KtoR-WW C-trimer remaining after 24 hours was determined by HPLC by comparing the peak areas. Peptide TF-18-KtoR-WW C-trimer in the form of L-amino acids was used as a control. Peptide TF-18-KtoR-WW C-trimer was stable under these conditions, with only slight degradation after 24 hours, while peptide TF-18-KtoR-WW C-trimer in the form of L-amino acids was essentially degraded in SGF and SIF after 8 hours or 1 hour, respectively.

[0451] Example 42. Whole blood cytokine release assay.

[0452] The effect of peptide TF-18-KtoR-WW C-trimer on IL-8 release in ex vivo stimulated human whole blood was evaluated. Three stimulators were included in the study: recombinant human TNF (Cell Systems #CS-C1140), LPS (Invivogen #REP-HEBS-10) and anti-CD3 plus anti-CD28 antibodies (ThermoFisher #16-0037-81 and #16-0289-81).

[0453] Whole blood analysis was performed under sterile conditions in a laminar flow hood. All conditions were tested in triplicate. Fresh human whole blood from three healthy donors (<4 hours after sampling, French National Blood Service Center) was anticoagulated with heparin.

[0454] The following controls were included in the assay: unstimulated blood; unstimulated blood in the presence of 100 nM of the peptide TF-18-KtoR-WW C-trimer; or TNF or LPS or anti-CD3 plus anti-CD28 antibody-stimulated blood in the absence of compound. Whole blood stimulation was performed in U-bottom 96-well plates as follows. For TNF stimulation, the peptide TF-18-KtoR-WW C-trimer and TNF (10 ng / mL) were pre-incubated at room temperature for 30 minutes before addition to diluted blood and further incubation at 37 °C, 5% CO2for 24 hours. For stimulation with LPS (0.1 EU / mL) or anti-CD3 plus anti-CD28 antibodies (0.1 pg / mL anti-CD3 and 1 pg / mL anti-CD28), the peptide TF-18-KtoR-WW C-trimer was mixed with diluted blood before the stimulator was added according to the plate layout. Control groups were prepared in a similar manner. Blood samples were incubated in RPMI containing stabilized L-glutamine at 37 °C, 5% CO2. After 24 hours of incubation at 37 °C, 5% CO2, supernatants were collected and stored at -20 °C. The final reaction volume was 300 pL per well with a final blood concentration of 50%. Released IL-8 in culture supernatants was measured using a specific ELISA kit and according to the supplier’s instructions (Human IL-8 ELISA Kit, R&D Systems, #DY208).

[0455] No signs of hemolysis were observed in the collected supernatants after 24 hours of incubation. IL-8 levels averaged between 450 and 1070 pg / mL in the absence of stimulation. Incubation of blood with 100 nM of the peptide TF-18-KtoR-WW C-trimer alone did not induce IL-8. In the presence of 100 nM of the peptide TF-18-KtoR-WW C-trimer, a decrease in spontaneous IL-8 release was observed. In the presence of the peptide TF-18-KtoR-WW C-trimer, a clear dose-dependent decrease in IL-8 release by stimulated blood was observed in three donors. The degree of inhibition of IL-8 by the peptide TF-18-KtoR-WW C-trimer varied between the three stimulators. TNF-induced IL-8 was completely inhibited by 100 nM (10 nM for donor 2) of the peptide TF-18-KtoR-WW C-trimer. LPS-induced IL-8 was partially inhibited in the blood of donors 1 and 3, but was not modulated in the blood of donor 2. Anti-CD3 plus anti-CD28 antibody-induced IL-8 was also completely (donors 1 and 3) or partially (donor 2) inhibited in the presence of the peptide TF-18-KtoR-WW C-trimer. See Figure 2, which shows the average IL-8 inhibition rate for three samples under each condition. Figure 25

[0456] ​Example 43. Peptide TF-18-KtoR-WW C-trimer promotes in vivo survival in a human TNF alpha mouse challenge model.

[0457] At time zero (T=0), healthy normal mice were challenged with LD 80 of human TNF alpha (HuTNF alpha) and monitored for 24 hours. Thirty minutes prior to challenge, mice received pretreatment with anti-TNF alpha monoclonal antibody (study grade Adalimumab; R&D Systems) or peptide TF-18-KtoR-WW C-trimer at a dose of 1X or 5X the challenge dose of huTNF alpha. At 0.5 hours and 1.5 hours, mice were again treated with peptide TF-18-KtoR-WW C-trimer. See Figure 26 , anti-TNF alpha monoclonal antibody (anti-TNF) inhibits both mouse and human TNF-alpha, while peptide TF-18-KtoR-WW C-trimer inhibits only human TNF-alpha.

[0458] Example 44. Single dose subcutaneous administration of peptide TF-18-KtoR-WW C-trimer

[0459] Mice were administered peptide TF-18-KtoR-WW C-trimer diluted in PBS in a single subcutaneous dose to 2 male and 2 female CD1 mice. See Figure 27 and Figure 28 , showing the average amount of peptide TF-18-KtoR-WW C-trimer in the plasma of 4 animals (2 male and 2 female) per group. Figures A and B present two different graphical representations of the data.

[0460] Example 45. Plasma levels of peptide TF-18-KtoR-WW C-trimer following oral administration

[0461] Peptide TF-18-KtoR-WW C-trimer was diluted to 50 mg / kg with PBS and administered to mice, 2 male and 2 female mice, via a single oral gavage prior to serum collection. See Table 1 below for the concentration (ng / mL) of peptide TF-18-KtoR-WW C-trimer in the serum of all four animals at 6, 12, and 24 hour time points post-dose. Peptide TF-18-KtoR-WW C-trimer was detectable in the plasma of all four animals 24 hours post-oral administration. Table 1. Serum concentration of peptide TF-18-KtoR-WW C-trimer following oral administration Time Male 1 Male 2 Female 1 Female 2 6h 0 0 0 0 12h 0 0 15.9 0 24h 77.5 149 15.9 149

[0462] Example 46. Oral administration of peptide TF-18-KtoR-WW C-trimer

[0463] Peptide TF-18-KtoR-WW C-trimer was administered to C57B1 / 6 mice (3 animals per group) intravenously (IV; 5 mg / kg), subcutaneously (SC; 50 mg / kg) and orally (po; 100 mg / kg). The vehicle was 91% sodium phosphate buffer (20 mM) and 9% sucrose. The concentration (nM) of peptide TF-18-KtoR-WW C-trimer in plasma or kidney (in case of the 50 mg / kg SC dose) was measured by mass spectrometry. Briefly, the luminal contents of the intestinal, liver and kidney tissues were thoroughly rinsed before homogenization and analysis. Peptide TF-18-KtoR-WW C-trimer was extracted from serum by precipitation of serum components using an organic solvent (crash acetonitrile), separated from remaining contaminating proteins using C18 reverse phase HPLC at 40°C and identified using ESI-MS. The level of peptide TF-18-KtoR-WW C-trimer was quantified by comparing the signal of the unknown sample to a standard curve and quality control samples. All samples were spiked with a known amount of a "heavy" internal standard to control variability in the extraction, injection and ionization processes.

[0464] See Table 2 and Figure 29 Peptide TF-18-KtoR-WW C-trimer was detected in plasma after IV administration of 5.0 mg / kg or SC administration of 50 mg / kg. It was also detected in the kidney after SC dosing of 50 mg / kg; 24 hours later, 10% of the initial exposure amount was present in the kidney. The long terminal half-life of peptide TF-18-KtoR-WW C-trimer was 18 hours after intravenous administration.

[0465] See Table 3 and Figure 30 Oral (PO) dosing (100 mg / kg) resulted in detectable amounts of peptide TF-18-KtoR-WW C-trimer in the liver and kidney (2 out of 3 animals), indicating that some peptide TF-18-KtoR-WW C-trimer entered systemic circulation. Plasma exposure was detectable in 1 animal per group. Peptide TF-18-KtoR-WW C-trimer was detectable in the liver and kidney, indicating systemic distribution after oral delivery. In addition, high concentrations of peptide TF-18-KtoR-WW C-trimer were observed in both small and large intestinal tissue. Table 2. Pharmacokinetics after 5.0 mg / kg IV and 50 mg / mg SC dosing 50 mg / kg IV 50 mg / mg SC Cl (mL / min / kg) 2.5 V SS (L / kg) 1.1 Half-life (h) 18 MRT (h) 7.5 AUC LAST (nM·h)]]> 3,82018,100 AUC TOTAL (nM·)]]> 4,30018,200 AUC extra (%) 110.9 C MAX (nM) 6,680 T MAX (h)]]> 0.5 F SC (%)]] 47 Table 3. Pharmacokinetics after 100 mg / kg oral dosing

[0466] Example 47. LPS challenge model

[0467] Tg1278 (mTNF KO / hTNF KI) mice are a transgenic strain that normally regulate and express human TNF alpha (hTNF alpha) in the absence of murine TNF alpha. To induce acute TNF-mediated inflammation, groups of male and female mice received a single injection of 10 μg / mouse of LPS. Six hours after LPS treatment, mouse sera were collected for analysis of IL-6 production. IL-6 was detected by ELISA. See Figure 31 At a dose of 5, 15 and 50 mg / kg / mouse of the peptide TF-18-KtoR-WW C-trimer administered subcutaneously one hour prior to LPS treatment, the results were complete inhibition of TNF-mediated IL-6 production at all doses. The activity of the peptide TF-18-KtoR-WW C-trimer was equivalent to that of 10 mg / kg / mouse of the study drug entanercept (labeled Enbrel in the graph) administered 16 hours prior to LPS treatment. See Figure 32 The peptide TF-18-KtoR-WW C-trimer was able to inhibit mouse KC (Chemokine (C-X-C motif) ligand 1 (CXCL1)) production in a similar manner.

[0468] Various publications, including patents, patent application publications and scientific articles, are cited throughout this application, the disclosures of which are incorporated by reference in their entireties for all purposes.

[0469] While exemplary embodiments have been shown and described, it is to be understood that various other modifications can be made to them without departing from the spirit and scope of the application. Table 4 Peptide sequences disclosed SEQ ID NO: 1 C-X 2 -X 3 -X 4 -X 5 -X 6 -F-F-N-X 10 -X 11 -C, wherein X 2 to X 6 , X 10 each is a D-amino acid or a D-alpha-amino acid analog thereof; and X 2 is any one of Thr (T), Val (V), His (H), Leu (L), Gin (Q), Ala (A), He (I), Met (M), or Trp (W) in the D form, or a D-alpha-amino acid analog thereof; X 3is a D-form Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), or Leu (L), or a D- alpha-amino acid analog thereof; X 4 is a polar amino acid including a D-form Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gin (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G), or a D-alpha-amino acid analog thereof; X 5 is a D-form Pro, Trp, His, Gin, Gly, Arg, Ala, Val, or Leu, or a D-alpha-amino acid thereof; X 6 is a D-form Arg (R), His (H), Lys (K), Glu (E), Gin (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-alpha-amino acid analog thereof; X 10 is a D-form Trp (W) or Tyr (Y), or a D-alpha-amino acid analog thereof; X 11 is a D-form Trp (W), Tyr (Y), Gin (Q), or His (H), or a D-alpha-amino acid analog thereof; and C represents a D-form cysteine, F represents a D-form phenylalanine, and N represents a D-form asparagine, or a D-alpha-amino acid analog of C, F, or N. SEQ ID NO: 2 C*-X 2 -X 3 -X 4 -X 5 -X 6 -F-N-N-X 10 -X 11 -C*(SEQ ID NO: 2), wherein X 1 to X 6 , X 10 and X 11 each are a D-amino acid or a D-alpha-amino acid analog thereof, and C, F, and N are D-form cysteine, phenylalanine, and asparagine, or a D-alpha-amino acid analog thereof; and X 2 is a D-form Thr (T), Val (V), His (H), Leu (L), Gin (Q), Ala (A), He (I), Met (M), or Trp (W), or a D-alpha-amino acid analog thereof; X 3selected from D-form Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), or Leu (L), or a D-a-amino acid analog thereof; X 4 is a polar amino acid selected from D-form Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gin (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G), or a D-a-amino acid analog thereof; X 5 is D-form Pro (P), Trp (W), His (H), Gin (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), or a D-a-amino acid analog thereof; X 6 is D-form Arg (R), His (H), Lys (K), Glu (E), Gin (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-a-amino acid analog thereof; X 10 is D-form Trp (W) or Tyr (Y), or a D-a-amino acid analog thereof; X 11 is D-form Trp (W), Tyr (Y), Gin (Q), or His (H), or a D-a-amino acid analog thereof; and * represents an optional intramolecular disulfide bond, C represents D-form cysteine or a D-a-amino acid analog thereof, F represents D-form phenylalanine or a D-a-amino acid analog thereof; and N represents D-form asparagine or a D-a-amino acid analog thereof. SEQ ID NO: 3 C*-X 2 -[W / F / Y]-X 4 -X 5 -X 6 -F-N-N-[W / Y]-W-C* (SEQ ID NO: 3) X 2 is D-form Thr (T), Val (V), His (H), Leu (L), Gin (Q), Ala (A), He (I), Met (M), or Trp (W), or a D-a-amino acid analog thereof; X 4 is a polar amino acid selected from D-form Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gin (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G), or a D-a-amino acid analog thereof; X 5 is D-form Pro (P), Trp (W), His (H), Gin (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), or a D-a-amino acid analog thereof; X 6 is D-form Arg (R), His (H), Lys (K), Glu (E), Gin (Q), Val (V), Leu (L), Ser (S), or Ala (A) or a D-a-amino acid analog thereof; and * represents an optional intramolecular disulfide bond, C represents D-form cysteine or a D-a-amino acid analog thereof, F represents D-form phenylalanine or a D-a-amino acid analog thereof; W represents D-form tryptophan or a D-a-amino acid analog thereof, F- represents D-form phenylalanine or a D-a-amino acid analog thereof, Y- represents D-form tyrosine or a D-a-amino acid analog thereof, and N represents D-form asparagine or a D-a-amino acid analog thereof. SEQ ID NO: 4 CVWQPKFNNYWC SEQ ID NO: 5 CVWQPRFNNYWC SEQ ID NO: 6 CTFQPRFNNYWC SEQ ID NO: 7 CTFQPRFNNWWC SEQ ID NO: 8 CSFQPRFNNYWC SEQ ID NO: 9 CSFQPRFNNWWC SEQ ID NO: 10 CVFQPRFNNYWC SEQ ID NO: 11 CVFQPRFNNWWC SEQ ID NO: 12 CTFQWRFNNYWC SEQ ID NO: 13 CLYQPVFNNWWC SEQ ID NO: 14 CVFQAAFNNYWC SEQ ID NO: 15 CVFQHHFNNWWC SEQ ID NO: 16 CHFNPRFNNWWC SEQ ID NO: 17 CVWQPHFNNYWC SEQ ID NO: 18 CVFQGRFNNWWC SEQ ID NO: 19 CVFQHRFNNWWC SEQ ID NO: 20 CVFNPRFNNWWC SEQ ID NO: 21 CVFKPRFNNWWC SEQ ID NO: 22 CAYQRQFNNWWC SEQ ID NO: 23 CWFEHRFNNWHC SEQ ID NO: 24 CHFQHRFNNWWC SEQ ID NO: 25 CHFQPRFNNWWC SEQ ID NO: 26 CTYQPRFNNWWC SEQ ID NO: 27 CQFQPRFNNWQC SEQ ID NO: 28 CHFSQRFNNWWC SEQ ID NO: 29 VRSSSRT PSDKPVAHVV ANPQAEGQLQ WLNRRANALL ANGVELRDNQLVVPSEGLYLIYSQVLFKGQ GCPSTHVLLT HTISRIA VSY QTKVNLLSAI KSPCQRETPE GAEAKPWYEPIYLGGVFQLE KGDRLSAEIN RPDYLDFAES GQVYFGIIAL SEQ ID NO: 30 GGEEK SEQ ID NO: 31 GGRRRK SEQ ID NO: 32 - Peptide DD-18 DDCVWQPKFNNYWC SEQ ID NO: 33 - Peptide 18V1 CVWQPKFNNYWCGGEEEK SEQ ID NO: 34 - Peptide 18V2 CVWQPKFNNYWCGGRRRK SEQ ID NO: 35 - Peptide DD-18 mutant DDCVWQPKFNNYGC SEQ ID NO: 36 - Peptide DD-18 di-mutant DDCDWQDKFNNYWC SEQ ID NO: 37 - Peptide DD-18-ex DDCVWQPKFNNYWCGGSAETVE SEQ ID NO: 38 - Peptide DGA-18 DGACVWQPKFNNYWC SEQ ID NO: 39 - Peptide DD-18-KtoR DDCVWQPRFNNYWC SEQ ID NO: 40 - Peptide DD-18-KtoR-R3 DDCVWQPRFNNYWCGGRRRK SEQ ID NO: 41 - Peptide DD-Hit16 DDCHFNPRFNNWWC SEQ ID NO: 42 - Peptide DD-18-noVal DDCWQPKFNNYWC SEQ ID NO: 43 - Peptide DD-18-plusAla DDCAVWQPKFNNYWC SEQ ID NO: 44 - Peptide DD-18-KtoR-PEG12-K DDCVWQPRFNNYWC-PEG12-K SEQ ID NO: 45 Peptide K-PEG12-DD-18-KtoR K-PEG12-DDCVWQPRFNNYWC SEQ ID NO: 46 - IZ coiled coil IKKEIEAIKKEQEAIKKKIEAIEKEA SEQ ID NO: 47 - Peptide 018-WtoG DDCVGQPKFNNYWC SEQ ID NO: 48 - Peptide 018-NtoG DDCVWQPKFNGYWC SEQ ID NO: 49 - Peptide DDD-018 DDDCVWQPKFNNYWC SEQ ID NO: 50 - Peptide DD-hitl9 DDCQFQPRFNNWQC SEQ ID NO: 51 - Peptide DD-hit33 DDCHFSQRFNNWWC SEQ ID NO: 52 - Peptide DD-hit92 DDCAYQRQFNNWWC SEQ ID NO: 53 - Peptide DD-hitl93 DDCWFEHRFNNWHC SEQ ID NO: 54 - PEG4-DD-18 PEG4-DDCVWQPKFNNYWC - Amide SEQ ID NO: 55 - DD-PEG4-018 DD-PEG4-CVWQPKFNNYWC SEQ ID NO: 56 - Peptide D-018 DCVWQPKFNNYWC SEQ ID NO: 57 - Peptide DD-018-NtoQ DDCVWQPKFNQYWC SEQ ID NO: 58 - Peptide DD-TF-018-KtoR DDCTFQPRFNNYWC SEQ ID NO: 59 - Peptide DD-Hitl4 DDCVFQHHFNNWWC SEQ ID NO: 60 - Peptide DDD-018-KtoR DDDCVWQPRFNNYWC SEQ ID NO: 61 - Peptide DD-TF-018-KtoR-WW DDCTFQPRFNNWWC SEQ ID NO: 62 - Peptide DD-VF-018-KtoR DDCVFQPRFNNYWC SEQ ID NO: 63 - Peptide DD-SF-018-KtoR DDCSFQPRFNNYWC SEQ ID NO: 64 - VF-018 - KtoR - WW DDCVFQPRFNNWWC SEQ ID NO: 65 - DtoE - VF-018 - KtoR - WW EDCVFQPRFNNWWC SEQ ID NO: 66 - VF-018 - QtoK - KtoR - WW DDCVFKPRFNNWWC SEQ ID NO: 67 - VF-018 - QtoKbio - KtoR - WW DDCVFX 6 PRFNNWWC; wherein X 6 is K (biotin) SEQ ID NO: 68 - VF-018 - PtoG - KtoR - WW DDCVFQGRFNNWWC SEQ ID NO: 69 - VF-018 - PtoH - KtoR - WW DDCVFQHRFNNWWC SEQ ID NO: 70 - VF-018 - QtoN - KtoR - WW DDCVFNPRFNNWWC SEQ ID NO: 71 - sucD-018 - KtoR suc-DCVWQPRFNNYWC, wherein suc is succinic acid SEQ ID NO: 72 - Pen-TF-018 - KtoR - WW DDXTFQPRFNNWWC, wherein X is D-penicillamine SEQ ID NO: 73 - TF-018 - KtoR - WW - Pen DDCTFQPRFNNWWX, wherein X is D-penicillamine SEQ ID NO: 74 - homoLeu-F-018 - KtoR - WW DDCXFQPRFNNWWC, wherein X is D-homoleucine SEQ ID NO: 75 - norLeu-F-018 - KtoR - WW DDCXFQPRFNNWWC, wherein X is D-norleucine SEQ ID NO: 76 - DD-018 - WtoG2 DDCVGQPKFNNYGC SEQ ID NO: 77 CIFQQQFNNYWC SEQ ID NO: 78 CMHQQRFNNWWC SEQ ID NO: 79 CTFVRFNNYWC SEQ ID NO: 80 CIFQWRFNNYWC SEQ ID NO: 81 CTFQWHFNNYWC SEQ ID NO: 82 CVFQHLFNNWWC SEQ ID NO: 83 CTFQWLFNNYWC SEQ ID NO: 84 CAFQWRFNNYWC SEQ ID NO: 85 CTFRWRFNNYWC SEQ ID NO: 86 CTSQWRFNNYWC SEQ ID NO: 87 CTFQLRFNNYWC SEQ ID NO: 88 CTFQVRFNNYWC SEQ ID NO: 89 CIWQPKFNNYWC SEQ ID NO: 90 CTFQRRFNNYWC SEQ ID NO: 91 CTFQWSFNNYWC SEQ ID NO: 92 CVFQRHFNNWWC SEQ ID NO: 93 CVSTHHFNNWWC SEQ ID NO: 94 CAFQHHFNNWWC SEQ ID NO: 95 CAYQRHFNNWWC SEQ ID NO: 96 CHFNPLFNNWWC SEQ ID NO: 97 CHFNRRFNNWWC SEQ ID NO: 98 CHFSQLFNNWWC SEQ ID NO: 99 CLYQLVFNNWWC SEQ ID NO: 100 CQFRPRFNNWQC SEQ ID NO: 101 CTLQQQFNNYWC SEQ ID NO: 102 CTSRWRFNNYWC SEQ ID NO: 103 CVFQASFNNYWC SEQ ID NO: 104 CVFQHSFNNWWC SEQ ID NO: 105 CVFRHHFNNWWC SEQ ID NO: 106 CVFTHHFNNWWC SEQ ID NO: 107 CVSQHHFNNWWC SEQ ID NO: 108 CVWQPEFNNYWC SEQ ID NO: 109 CVWQQKFNNYWC SEQ ID NO: 110 CVWRPKFNNYWC SEQ ID NO: 111 C-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -C, wherein each of X 2 to X 11 is a D-amino acid; and X 2is the D-form of any typical L-amino acid except cysteine; X 3 is D-form Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), or Leu (L); X 4 is a polar amino acid including Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gln (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G); X 5 is the D-form of any typical L-amino acid except cysteine; X 6 is the D-form of any typical L-amino acid except cysteine; X 7 is D-form Phe (F); X 8 is D-form Asn (N), X 9 is D-form Asn (N), X 10 is D-form Trp (W) or Tyr (Y); X 11 is D-form Trp (W), Gln (Q), Tyr, or His (H); and C represents D-form cysteine. SEQ ID NO: 112 C*-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -C*, wherein X 2 to X 11 each of which is a D-amino acid; and X 2 is the D-form of any typical L-amino acid except cysteine; X 3 is D-form Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), and Leu (L); X 4 is a polar amino acid including Arg (R), Lys (K), His (H), Glu (E), Asp (D), Gln (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), and Gly (G); X 5 is the D-form of any typical L-amino acid except cysteine; X 6 is the D-form of any typical L-amino acid except cysteine; X 7 is D-form Phe (F); X 8 is D-form Asn (N), X 9is D-Asn (N), X 10 is D-Trp (W) and Tyr (Y); X 11 is D-Trp (W), Tyr (Y), Gin (Q) or His (H); C represents D-cysteine; and * represents an optional intramolecular bond. SEQ ID NO: 113 C*-X 2 -[W / F / Y]-[Polar]-X 5 -X 6 -F-N-N-[W / Y]-W-C* (SEQ ID NO: 3), wherein X 2 is the D-form of any typical L-amino acid except Cys, X 5 is the D-form of any typical L-amino acid except Cys, X 6 is the D-form of any typical L-amino acid except Cys, Polar represents a D-amino acid comprising one of R, K, H, E, D, Q, N, T, S, P, A or G, and * represents an optional intramolecular disulfide bond. SEQ ID NO: 114 X*TFQPRFNNWWC*, wherein * represents an optional intramolecular disulfide bond, and X is D-penicillamine SEQ ID NO: 115 C*TFQPRFNNWX*, wherein * represents an optional intramolecular disulfide bond, and X is D-penicillamine

Claims

1. A D-peptide or a salt thereof, wherein the peptide comprises a core TNFα-binding domain of a D-amino acid and has the following amino acid sequence: CX 2 -X 3 -X 4 -X 5 -X 6 -FNNX 10- X 11 -C(SEQ ID NO: 1), where X 1 To X 6 X 10 and X 11 Each of them is a D-amino acid or its D-α-amino acid analogue; and aX 2 It is D-type Thr(T), Val(V), His(H), Leu(L), Gln(Q), Ala(A), Ile(I), Met(M) or Trp(W), or their D-α-amino acid analogs; bX 3 It is D-type Trp(W), Phe(F), Tyr(Y), Ser(S), His(H) and Leu(L), or their D-α-amino acid analogs; cX 4 These are polar amino acids, including D-type Arg(R), Lys(K), His(H), Glu(E), Asp(D), Gln(Q), Asn(N), Thr(T), Ser(S), Pro(P), Lys, Ala(A), and Gly(G), or their D-α-amino acid analogs; dX 5 It is D-type Pro(P), Trp(W), His(H), Gln(Q), Arg(R), Ala(A), Val(V), Leu(L) or Gly(G), or their D-α-amino acid analogs; eX 6 It is D-type Arg(R), His(H), Lys(K), Glu(E), Gln(Q), Val(V), Leu(L), Ser(S) or Ala(A), or their D-α-amino acid analogs; fX 10 It is D-type Trp(W) or Tyr(Y), or their D-α-amino acid analogs; gX 11 It is a D-type Trp(W), Tyr(Y), Gln(Q), or His(H), or a D-α-amino acid analog thereof; and kC represents D-cysteine ​​or its D-α-amino acid analogue; N represents D-asparagine or its D-α-amino acid analogue; and F represents D-phenylalanine or its D-α-amino acid analogue.

2. The D-peptide or its salt according to claim 1, wherein the core TNFα binding domain has the following amino acid sequence: CX 2 -[W / F / Y]-X 4 -X 5 -X 6 -FNN-[W / Y]-WC (SEQ ID NO: 3).

3. The D-peptide or its salt according to any one of claims 1 to 2, further comprising an intramolecular disulfide bond between cysteine ​​residues 1 and 12 of the core TNFα binding domain.

4. The D-peptide or a salt thereof according to any one of claims 1 to 3, wherein: aX 2 It is D-type Thr, Val, His, Leu, Gln, Ala, Ile, Met or Trp, or their D-α-amino acid analogs; bX 2 It is D-type Thr, Val, His, Leu or Gln, or their D-α-amino acid analogs; cX 2 It is a D-type Thr, Val, His, or Leu, or a D-α-amino acid analogue thereof; dX 2 It is D-type Thr, Val, or His, or their D-α-amino acid analogs; eX 2 It is D-type Thr or Val, or its D-α-amino acid analogue; fX 2 It is D-type Thr, or its D-α-amino acid analogue; or gX 2 It is D-type Val, or its D-α-amino acid analogue.

5. The D-peptide or a salt thereof according to any one of claims 1 or 3 to 4, wherein: aX 3 It is D-type Trp, Phe, Tyr or Ser, or their D-α-amino acid analogs; bX 3 It is D-type Trp, Phe, or Tyr, or their D-α-amino acid analogs; cX 3 It is D-type Trp or Phe, or their D-α-amino acid analogs; dX 3 It is D-type Trp, or its D-α-amino acid analogue; or eX 3 It is D-type Phe, or its D-α-amino acid analogue.

6. The D-peptide or a salt thereof according to any one of claims 1 to 5, wherein: aX 4 It is D-type Arg, His, Gln, Asn, Lys, Thr or Ser, or their D-α-amino acid analogs; bX 4 It is D-type Arg, His, Gln or Asn, or their D-α-amino acid analogs; cX 4 It is D-type Arg, His or Gln, or their D-α-amino acid analogs; dX 4 It is D-type Arg, Gln, or Asn, or their D-α-amino acid analogs; or eX 4 It is D-type Gln, or its D-α-amino acid analogue.

7. The D-peptide or a salt thereof according to any one of claims 1 to 6, wherein: aX 5 It is D-type Pro, Trp, His, Gln, Gly, Arg, Ala, Val, or Leu, or their D-α-amino acid analogs; bX 5 It is D-type Pro, Trp, His, Gln, Gly, Arg or Val, or their D-α-amino acid analogs; cX 5 It is D-type Pro, Trp, or His, or their D-α-amino acid analogs; dX 5 It is D-type Pro or Trp, or their D-α-amino acid analogs; or eX 5 It is D-type Pro, or its D-α-amino acid analogue.

8. The D-peptide or a salt thereof according to any one of claims 1 to 7, wherein: aX 6 It is D-type Arg, His, Lys, Glu, Gln, Val, Leu, Ser or Ala, or their D-α-amino acid analogs; bX 6 It is D-type Arg, His, Lys, Glu, Gln, Val or Leu, or their D-α-amino acid analogs; cX 6 It is D-type Arg, His, Lys, Glu or Gln, or their D-α-amino acid analogs; dX 6 It is a D-type Arg, His, Lys or Glu, or its D-α-amino acid analogue; eX 6 It is D-type Arg, Lys, or His, or their D-α-amino acid analogs; fX 6 It is D-type Arg, or its D-α-amino acid analogue; gX 6 It is D-type Lys, or its D-α-amino acid analogue, or hX 6 It is D-type His, or its D-α-amino acid analogue.

9. The D-peptide or a salt thereof according to any one of claims 1 to 8, wherein: aX 10 It is D-type Trp, or its D-α-amino acid analogue; or bX 10 It is D-type Tyr, or its D-α-amino acid analogue.

10. The D-peptide or a salt thereof according to any one of claims 1 to 9, wherein: aX 11 It is a D-type His(H), Trp(W), Tyr(Y) or Gln(Q), or its D-α-amino acid analogue; bX 11 It is D-type Tyr(Y), or its D-α-amino acid analogue, or cX 11 It is D-type Trp(W), or its D-α-amino acid analogue.

11. The D-peptide or a salt thereof according to any one of claims 1 to 10, wherein X 1 To X 6 X 10 and X 11 Each one in it is a D-amino acid.

12. The D-peptide or a salt thereof according to any one of claims 1 to 11, wherein the core TNFα binding domain has the following amino acid sequence: a.CVWQPKFNNYWC(SEQ ID NO: 4); b.CVWQPRFNNYWC (SEQ ID NO: 5); c.CTFQPRFNNYWC (SEQ ID NO: 6); d.CTFQPRFNNWWC(SEQ ID NO:7); e.CSFQPRFNNYWC (SEQ ID NO: 8); f.CSFQPRFNNWWC (SEQ ID NO: 9); g.CVFQPRFNNYWC (SEQ ID NO: 10); h.CVFQPRFNNWWC (SEQ ID NO: 11); i.CTFQWRFNNYWC(SEQ ID NO:12); j.CLYQPVFNNWWC (SEQ ID NO: 13); k.CVFQAAFNNYWC (SEQ ID NO: 14); l.CVFQHHFNNWWC (SEQ ID NO: 15); m.CHFNPRFNNWWC(SEQ ID NO:16); n.CVWQPHFNNYWC(SEQ ID NO:17); o.CVFQGRFNNWWC(SEQ ID NO: 18); p.CVFQHRFNNWWC (SEQ ID NO: 19); q.CVFNPRFNNWWC (SEQ ID NO: 20); r.CVFKPRFNNWWC(SEQ ID NO:21); s.CAYQRQFNNWWC(SEQ ID NO:22); t.CWFEHRFNNWHC (SEQ ID NO: 23); u.CHFQHRFNNWWC(SEQ ID NO:24); v.CHFQPRFNNWWC(SEQ ID NO:25); w.CTYQPRFNNWWC(SEQ ID NO:26); x.CQFQPRFNNWQC(SEQ ID NO: 27); or y.CHFSQRFNNWWC (SEQ ID NO: 28).

13. The D-peptide or a salt thereof according to any one of the preceding claims, wherein the core TNFα binding domain has the amino acid sequence shown in SEQ ID NO: 77 to 110.

14. The D-peptide or a salt thereof according to any one of the preceding claims, further comprising a tag sequence attached to the N-terminus of the peptide.

15. The D-peptide or a salt thereof according to claim 14, wherein the tag comprises the amino acid sequence D-Asp or D-AspAsp(DD).

16. The D-peptide or a salt thereof according to any one of the preceding claims, further comprising a tag sequence attached to the C-terminus of the peptide.

17. The D-peptide or a salt thereof according to claim 16, wherein the tag comprises the amino acid sequence D-GGEEEK (SEQ ID NO: 30) or D-GGRRRK (SEQ ID NO: 31).

18. The D-peptide or a salt thereof according to any one of the preceding claims, wherein the N-terminus of the peptide comprises a cap.

19. The D-peptide or a salt thereof according to claim 18, wherein the cap comprises an acetyl group or a protecting group.

20. A D-peptide or a salt thereof according to any one of the preceding claims, wherein the C-terminus of the peptide comprises a cap.

21. The D-peptide or a salt thereof according to claim 20, wherein the cap comprises an amide group or a protecting group.

22. The D-peptide or a salt thereof according to any one of the preceding claims, further comprising a polyethylene glycol (PEG) group.

23. The D-peptide or a salt thereof according to any one of the preceding claims, further comprising a linker.

24. The D-peptide or a salt thereof according to claim 23, wherein the linker comprises a PEG group.

25. The D-peptide or a salt thereof according to any one of claims 22 or 24, wherein the PEG group is attached to the N-terminus of the D-peptide.

26. The D-peptide or a salt thereof according to any one of claims 22 or 24, wherein the PEG group is attached to the C-terminus of the D-peptide.

27. The D-peptide or a salt thereof according to any one of claims 22 and 24 to 26, wherein each PEG group is a PEG group having 1 to 48 subunits, 1 to 30 subunits, 1 to 24 subunits or 1 to 12 subunits.

28. The D-peptide or a salt thereof according to claim 27, wherein each PEG group is a PEG group having 6, 8, 10, or 12 subunits.

29. A polymer of the D-peptide or a salt thereof, as described in any of the preceding claims.

30. The polymer or salt thereof according to claim 29, wherein the polymer is a dimer.

31. The polymer or salt thereof according to claim 29, wherein the polymer is a trimer.

32. The polymer or a salt thereof according to any one of claims 29 to 31, further comprising a polymeric backbone linked to the D-peptide, optionally via a linker.

33. The polymer or a salt thereof according to claim 32, wherein the polymer backbone is a trimer.

34. The polymer or a salt thereof according to claim 32, wherein the polymer backbone is a tetramer.

35. The polymer or a salt thereof according to any one of claims 33 or 34, wherein the trifunctional crosslinking agent is tris(succinimino)aminotriacetate (TSAT), tris-succinimino(6-aminohexanoyl)aminotriacetate (LC-TSAT), Fmoc backbone (bis(2,5-dioxopyrrolidone-1-yl)4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-(((2,5-dioxopyrrolidone-1-yl)oxy)-3-oxopropyl)pimelic acid ester, Fmoc backbone having a PEG27 chain, cyclohexane backbone (tris(2,5-dioxopyrrolidone-1-yl)cyclohexane-1,3,5-tricarboxylic acid ester), nitro backbone (bis(2,5-dioxopyrrolidone-1-yl)cyclohexane-1,3,5-tricarboxylic acid ester), nitro backbone (bis(2,5-dioxopyrrolidone-1-yl)cyclohexane-1,3,5-tricarboxylic acid ester), or nitro backbone (bis(2,5-dioxopyrrolidone-1-yl)cyclohexane-1,3,5-tricarboxylic acid ester). PEG-1-yl)4-(3-((2,5-dioxopyrrolidone-1-yl)oxy)-3-oxopropyl)-4-nitropimercate, amine skeleton (bis(2,5-dioxopyrrolidone-1-yl)4-amino-4-(3-((2,5-dioxopyrrolidone-1-yl)oxy)-3-oxopropyl)pimercate), amine-PEG27 skeleton, cholesterol skeleton, heterotetrameric PEG skeleton based on 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propionic acid skeleton, polymeric skeleton based on 4-amino-4-(2-carboxyethyl)pimercate or 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propionic acid.

36. The polymer or a salt thereof according to claim 35, wherein the polymeric peptide construct has the following structure: a.Fmoc-[peptide-PEG12-K-amide]3; b.Fmoc-[peptide-PEG4-K-amide]3; c.Fmoc-[peptide-PEG8-K-amide]3; d.Fmoc-[Ac-K-PEG12-peptide-amide]3; e. [peptide-PEG6-K-amide]3-Fmoc; f. [peptide-PEG12-K-amide]3-PEG27-Fmoc; g. [peptide-PEG12-K-amide]3-Fmoc; h. [peptide-PEG12-K-amide]3-cyclohexane; i. [peptide-PEG12-K-amide]3-nitro; j. [peptide-PEG12-K-amide]3-PEG27-amine; k. [peptide-PEG12-K-amide]3-amine; l. [peptide-PEG12-K-amide]3-PEG27-biotin; or m.[peptide-PEG12-K-amide]3-PEG27-cholesterol; and wherein the peptide is a D-peptide.

37. The D-peptide or a salt thereof according to any one of claims 1 to 28, wherein it has at least one of the following amino acid sequences: a.DDCVWQPKFNNYWC (SEQ ID NO: 32); b.DGACVWQPKFNNYWC (SEQ ID NO: 38); c.DDCVWQPRFNNYWC (SEQ ID NO: 39); d.DDCVWQPRFNNYWCGGRRRK (SEQ ID NO: 40); e.DCVWQPKFNNYWC(SEQ ID NO:56); f.DDDCVWQPRFNNYWC(SEQ ID NO: 60); g.DDCTFQPRFNNWWC (SEQ ID NO: 61); h.DDCVFQPRFNNYWC (SEQ ID NO: 62); i.DDCSFQPRFNNYWC(SEQ ID NO: 63); or j. DDCVFQPRFNNWWC (SEQ ID NO: 64).

38. The polymer or a salt thereof according to any one of claims 29 to 36, comprising a D-peptide having at least one of the following amino acid sequences: - a.DDCVWQPKFNNYWC (SEQ ID NO: 32); b.DGACVWQPKFNNYWC (SEQ ID NO: 38); c.DDCVWQPRFNNYWC (SEQ ID NO: 39); d.DDCVWQPRFNNYWCGGRRRK (SEQ ID NO: 40); e.DCVWQPKFNNYWC(SEQ ID NO:56); f.DDDCVWQPRFNNYWC(SEQ ID NO: 60); g.DDCTFQPRFNNWWC (SEQ ID NO: 61); h.DDCVFQPRFNNYWC (SEQ ID NO: 62); i.DDCSFQPRFNNYWC(SEQ ID NO: 63); or j. DDCVFQPRFNNWWC (SEQ ID NO: 64).

39. A pharmaceutical composition comprising at least one D-peptide or multipeptide construct or a pharmaceutically acceptable salt thereof, as described in any one of the preceding claims, and at least one pharmaceutically acceptable excipient or carrier.

40. The pharmaceutical composition according to claim 39, which is formulated for parenteral administration.

41. The pharmaceutical composition according to claim 40, which is formulated for intravenous, intramuscular or subcutaneous administration.

42. The pharmaceutical composition according to claim 39, which is formulated for oral administration.

43. The pharmaceutical composition according to claim 39, which is formulated for topical application.

44. The pharmaceutical composition according to claim 43, which is formulated for topical application to the skin (dermis) or eyes.

45. The pharmaceutical composition according to claim 39, which is formulated for rectal administration.

46. ​​A lyophilized composition comprising at least one D-peptide or multipeptide construct or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 38, and a stabilizer.

47. The lyophilized composition of the pharmaceutical composition and stabilizer according to any one of claims 39 to 45.

48. A rehydration solution of the lyophilized composition according to any one of claims 46 or 47.

49. A method of treating TNFα-mediated diseases, comprising administering an effective amount of any one of claims 1 to 28 or 37, any one of claims 29 to 36 and 38, or any one of claims 39 to 45, or a pharmaceutically acceptable salt thereof.

50. The method of claim 49, wherein the TNFα-mediated disease is adult Crohn's disease, juvenile Crohn's disease, ulcerative colitis, plaque psoriasis, cutaneous lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, hidradenitis suppurativa, uveitis (middle, posterior, or panuveitis), or radionegative spondyloarthritis.

51. The method of claim 50, wherein the TNFα-mediated disease is inflammatory bowel disease.

52. The method of claim 51, wherein the inflammatory bowel disease is adult Crohn's disease, childhood Crohn's disease, or ulcerative colitis.

53. The method according to claim 51 or 52, wherein the administration is oral administration.

54. The method according to claim 51 or 52, wherein the application is rectal.

55. The method according to claim 51 or 52, wherein the application is parenteral administration.

56. The method of claim 49, wherein the TNFα-mediated disease is an inflammatory skin disease.

57. The method of claim 56, wherein the inflammatory skin disease is plaque psoriasis or cutaneous lupus.

58. The method according to claim 56 or 57, wherein the application is a local application.

59. The method according to claim 56 or 57, wherein the application is parenteral administration.

60. The method of claim 49, wherein the TNFα-mediated disease is an inflammatory disease, wherein the inflammatory disease is systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, or hidradenitis suppurativa.

61. The method of claim 60, wherein the application is parenteral.

62. The method according to claim 55, 59 or 61, wherein the parenteral administration is selected from intravenous, subcutaneous or intramuscular administration.

63. A method for reducing TNFα-mediated inflammation, comprising administering to a subject the D-peptide of any one of claims 1 to 38 or a multimeric peptide construct thereof or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 39 to 45.

64. A method of inhibiting TNFα, comprising administering to a subject the D-peptide of any one of claims 1 to 38 or a multipeptide construct thereof or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 39 to 45.

65. A method for reducing TNFα-mediated inflammatory responses, comprising administering to a subject the D-peptide of any one of claims 1 to 38 or a multimeric peptide construct thereof or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 39 to 45.

66. The method according to any one of claims 63 to 65, wherein the administration is by oral administration, parenteral administration, topical application (dermis), or rectal administration.

67. The method according to any one of claims 63 to 65, wherein the D-peptide or its multipeptide construct or its pharmaceutically acceptable salt is applied topically to reduce TNFα activity or inflammation or inflammatory response.

68. The method according to any one of claims 63 to 67, wherein the subject suffers from a TNFα-mediated disease.

69. The method of claim 68, wherein the TNFα-mediated disease is adult Crohn's disease, juvenile Crohn's disease, ulcerative colitis, plaque psoriasis, cutaneous lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, hidradenitis suppurativa, uveitis (middle, posterior, or panuveitis), or radionegative spondyloarthritis.

70. The D-peptide or its multimeric peptide construct or its pharmaceutically acceptable salt according to any one of claims 1 to 38, used as a medicine.

71. The D-peptide or its multimeric peptide construct or its pharmaceutically acceptable salt according to any one of claims 1 to 38, in a method of treating a subject by means of a therapy.

72. The D-peptide of claim 71, wherein the subject suffers from a TNFα-mediated disease.

73. The D-peptide according to claim 72, wherein the TNFα-mediated disease is adult Crohn's disease, juvenile Crohn's disease, ulcerative colitis, plaque psoriasis, cutaneous lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or juvenile idiopathic arthritis, hidradenitis suppurativa, uveitis (middle, posterior, or panuveitis), or radionegative spondyloarthritis.

74. A D-peptide or a salt thereof, wherein the peptide comprises a core TNFα-binding domain of a D-amino acid and has the following amino acid sequence, CX 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -C(SEQ ID NO: 111), where X 2 To X 11 Each of them is a D-amino acid; and X 2 It is the D form of any typical L-amino acid except cysteine; X 3 For type D: Trp(W), Phe(F), Tyr(Y), Ser(S), His(H), or Leu(L); X 4 These are polar amino acids, including Arg(R), Lys(K), His(H), Glu(E), Asp(D), Gln(Q), Asn(N), Thr(T), Ser(S), Pro(P), Ala(A), or Gly(G); X 5 It is the D form of any typical L-amino acid except cysteine; X 6 It is the D form of any typical L-amino acid except cysteine; X 7 Phe(F) of type D; X 8 For D-type Asn(N), X 9 For D-type Asn(N), X 10 For D-type Trp(W) or Tyr(Y); X 11 It can be D-type Trp(W), Gln(Q), Tyr, or His(H); and C represents D-type cysteine.

75. A D-peptide or a salt thereof, wherein the peptide comprises a core TNFα-binding domain of a D-amino acid and has the following amino acid sequence, C*-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -C*(SEQ ID NO: 112), where X 2 To X 11 Each of them is a D-amino acid; and X 2 It is the D form of any typical L-amino acid except cysteine; X 3 The D-type Trp(W), Phe(F), Tyr(Y), Ser(S), His(H), and Leu(L) are also included. 4 These are polar amino acids, including Arg(R), Lys(K), His(H), Glu(E), Asp(D), Gln(Q), Asn(N), Thr(T), Ser(S), Pro(P), Ala(A), and Gly(G); X 5 It is the D form of any typical L amino acid except cysteine; X 6 It is the D form of any typical L amino acid except cysteine; X 7 Phe(F) of type D; X 8 For D-type Asn(N), X 9 For D-type Asn(N), X 10 For D-type Trp(W) and Tyr(Y); X 11 It can be D-type Trp(W), Tyr(Y), Gln(Q), or His(H); C represents D-type cysteine; and * represents an optional intramolecular bond.

76. A D-peptide or a salt thereof, wherein the peptide comprises a core TNFα-binding domain of a D-amino acid and has the following amino acid sequence, C*-X 2 -[W / F / Y]-[Polar]-X 5 -X 6 -FNN-[W / Y]-WC*(SEQ ID NO: 113), where X 2 X is the D-type of any typical L-amino acid except Cys. 5 X is the D form of any typical L-amino acid except cysteine. 6 Polar represents the D-type of any typical L-amino acid other than cysteine. Polar indicates a D-amino acid containing one of R, K, H, E, D, Q, N, T, S, P, A, or G, and * indicates an optional intramolecular disulfide bond.

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