TNF-alpha binders and methods of using same

Abstract

D-peptide binders of TNFα, multimers thereof, and methods of using D-peptides and multimers thereof are provided. D-peptide-based inhibitors of TNFα are provided herein. 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. TNFi peptides contain a core TNFα-binding domain composed of D-amino acids.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 483,839, filed February 8, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] Sequence Listing Statement The Sequence Listing XML associated with this application is provided in XML format and is incorporated herein by reference. The name of the XML file containing the Sequence Listing is 4298-P1WO_Seq_List_20240207.xml. The XML file is 689,475 bytes; was created on February 7, 2024; and has been submitted electronically via the Patent Center along with the application herein.

[0003] STATEMENT OF GOVERNMENT LICENSE RIGHTS This invention was made with government support under SBIR grant project number 1R43DK117777-01 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0004] background FDA-approved anti-TNFα therapies include the four antibodies HUMIRA® (adalimumab), REMICADE® (infliximab), CIMZIA® (certolizumab pegol), and SIMPONI ARIA® (golimumab), as well as ENBREL® (etanercept), a soluble form of a TNFR. 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. The value of anti-TNFα therapy in treating chronic inflammatory diseases is clear, as HUMIRA® is the best-selling drug worldwide. However, approved anti-TNFα biologics are limited by the requirement for parenteral administration, increased systemic distribution and risk of infection associated with immunosuppression, and immunogenicity associated with anti-drug antibodies (ADAs), which result in loss of therapeutic activity. The present invention addresses this and other needs. Summary of the Invention [Means for solving the problem]

[0005] overview This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0006] 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. TNFi peptides contain a core TNFα-binding domain composed of D-amino acids.

[0007] In some embodiments, there is provided a D-peptide or salt thereof, wherein the peptide comprises a core TNFα binding domain of D-amino acids 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), and X 2 From X 11 each of which is a D-amino acid; 2 is the D-form of any of the common L-amino acids other than 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), Gln (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G); X 5 is the D-form of any of the common L-amino acids other than cysteine; X 6 is the D-form of any of the common L-amino acids other than cysteine; X 7 is the D form of Phe(F); X 8 is the D-type of Asn(N), and X 9 is the D-type of Asn(N), and X 10 is the D-form of Trp (W) or Tyr (Y); X 11 is the D-form of Trp (W), Gln (Q), Tyr, or His (H); and C represents the D-form of cysteine, thereby providing a D-peptide or a salt thereof.

[0008] In some embodiments, the peptide comprises a core TNFα binding domain of D-amino acids and has the following amino acid sequence: * -X 2 -X 3 -X 4 -X 5 -X 6 -X7 -X 8 -X 9 -X 10 -X 11 -C * (SEQ ID NO: 112), and X 2 From X 11 each of which is a D-amino acid; 2 is the D-form of any of the common L-amino acids other than cysteine; X 3 are the D forms of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), and Leu (L); X 4 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 is the D-form of any of the common L-amino acids other than cysteine; X 6 is the D-form of any of the common L-amino acids other than cysteine; X 7 is the D form of Phe(F); X 8 is the D-type of Asn(N), and X 9 is the D-type of Asn(N), and X 10 is the D-form of Trp (W) and Tyr (Y); X 11 is the D-form of Trp (W), Tyr (Y), Gln (Q) or His (H); C represents the D-form of cysteine; * represents an optional intramolecular bond, or a salt thereof.

[0009] In some embodiments, the peptide comprises a core TNFα binding domain of D-amino acids and has the following amino acid sequence: * -X 2 -[W / F / Y]-[Polarity]-X 5 -X 6 -FNN-[W / Y]-WC * (SEQ ID NO: 113), and X 2 is the D form of any of the standard L-amino acids other than Cys, and X 5is the D-form of any of the standard L-amino acids other than cysteine, and X 6 represents the D-amino acid, which is the D-form of any of the standard L-amino acids other than cysteine, and polarity includes one of R, K, H, E, D, Q, N, T, S, P, A, or G; * provides D-peptides or salts thereof, which optionally exhibit an intramolecular disulfide bond.

[0010] In some embodiments, there is provided a D-peptide or salt thereof, wherein the peptide comprises a core TNFα binding domain of D-amino acids 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), and X 2 From X 6 , X 10 each of which is a D-amino acid or a D-α-amino acid analog thereof; aX 2 is the D-form of any of Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), Ile (I), Met (M), or Trp (W), or a D-α-amino acid analog thereof; bX 3 is the D-form of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H) or Leu (L), or a D-α-amino acid analog thereof; cX 4 is a polar amino acid, including the D-form of 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-α-amino acid analog thereof; dX 5 is the D-form of Pro, Trp, His, Gln, Gly, Arg, Ala, Val, or Leu, or a D-α-amino acid thereof; eX 6is the D-form of Arg (R), His (H), Lys (K), Glu (E), Gln (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-α-amino acid analog thereof; fx 10 is the D-form of Trp (W) or Tyr (Y), or a D-α-amino acid analog thereof; gX 11 is the D-form of Trp (W), Tyr (Y), Gln (Q) or His (H), or a D-α-amino acid analog thereof; D-peptides or salts thereof are provided in which hC represents the D-form of cysteine, F represents the D-form of phenylalanine, and N represents the D-form of 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: * -X 2 -X 3 -X 4 -X 5 -X 6 -FNNX 10 -X 11 -C * (SEQ ID NO: 2), and X 1 From X6, X 10 and each of X11 is a D-amino acid; aX 2 is the D-form of Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), Ile (I), Met (M), or Trp (W), or a D-α-amino acid analog thereof; bX 3 is the D-form of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H) or Leu (L), or a D-α-amino acid analog thereof; cX 4is a polar amino acid, including the D-form of 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-α-amino acid analog thereof; dX 5 is the D-form of Pro (P), Trp (W), His (H), Gln (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), or a D-α-amino acid analog thereof; eX 6 is the D-form of Arg (R), His (H), Lys (K), Glu (E), Gln (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-α-amino acid analog thereof; fx 10 is the D-form of Trp (W) or Tyr (Y), or a D-α-amino acid analog thereof; gX 11 is the D-form of Trp (W), Tyr (Y), Gln (Q) or His (H); C represents the D-form of cysteine; F represents the D-form of phenylalanine; N represents the D-form of asparagine; * indicates an optional intramolecular disulfide bond.

[0012] In some embodiments, the D-peptide or salt thereof, 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), 2 is the D form of Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), Ile (I), Met (M), or Trp (W); X 4 is the D-form of 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 5is the D-form of Pro(P), Trp(W), His(H), Gln(Q), Arg(R), Ala(A), Val(V), Leu(L), or Gly(G), and X 6 is the D-form of Arg (R), His (H), Lys (K), Glu (E), Gln (Q), Val (V), Leu (L), Ser (S), or Ala (A); * provides a D-peptide or salt thereof, which exhibits an optional intramolecular disulfide bond between the indicated cysteine ​​residues.

[0013] In some embodiments, a D-peptide or a salt thereof is provided, further comprising an intramolecular disulfide bond between cysteine ​​residues of the core TNFα binding domain.

[0014] In some embodiments, a D-peptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or a salt thereof, aX 2 is the D-form of Thr, Val, His, Leu, Gln, Ala, Ile, Met, or Trp, or a D-α-amino acid analog thereof; bX 2 is the D-form of Thr, Val, His, Leu, or Gln, or a D-α-amino acid analog thereof; cX 2 is the D-form of Thr, Val, His, or Leu, or a D-α-amino acid analog thereof; dX 2 is the D-form of Thr, Val, or His, or a D-α-amino acid analog thereof; eX 2 is the D-form of Thr or Val, or a D-α-amino acid analogue thereof; fx 2 is the D-form of Thr, or a D-α-amino acid analog thereof; or gX 2 is the D-form of Val, or a D-α-amino acid analog thereof, or a salt thereof.

[0015] In some embodiments, a D-peptide having an amino acid sequence set forth in either SEQ ID NO: 1 or 2, or a salt thereof, aX 3 is the D-form of Trp, Phe, Tyr, or Ser, or a D-α-amino acid analog thereof; bX 3 is the D-form of Trp, Phe, or Tyr, or a D-α-amino acid analog thereof; cX 3 is the D-form of Trp or Phe, or a D-α-amino acid analog thereof; dX 3 is the D-form of Trp or a D-α-amino acid analog thereof; or eX 3 is the D-form of Phe, or a D-α-amino acid analog thereof, or a salt thereof.

[0016] In some embodiments, a D-peptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or a salt thereof, aX 4 is the D-form of Arg, His, Gln, Asn, Lys, Thr, or Ser, or a D-α-amino acid analog thereof; bX 4 is the D-form of Arg, His, Gln, or Asn, or a D-α-amino acid analog thereof; cX 4 is the D-form of Arg, His, or Gln, or a D-α-amino acid analog thereof; dX 4 is the D-form of Arg, Gln, or Asn, or a D-α-amino acid analog thereof; or eX 4 is the D-form of Gln, or a D-α-amino acid analog thereof, or a salt thereof.

[0017] In some embodiments, a D-peptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or a salt thereof, aX5 is the D-form of Pro, Trp, His, Gln, Gly, Arg, Ala, Val, or Leu, or a D-α-amino acid analog thereof; bX 5 is the D-form of Pro, Trp, His, Gln, Gly, Arg, or Val, or a D-α-amino acid analog thereof; cX 5 is the D-form of Pro, Trp, or His, or a D-α-amino acid analog thereof; dX 5 is the D-form of Pro or Trp, or a D-α-amino acid analog thereof; or eX 5 is the D-form of Pro, or a D-α-amino acid analog thereof, or a salt thereof.

[0018] In some embodiments, a D-peptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or a salt thereof, aX 6 is the D-form of Arg, His, Lys, Glu, Gln, Val, Leu, Ser, or Ala, or a D-α-amino acid analog thereof; bX 6 is the D-form of Arg, His, Lys, Glu, Gln, Val, or Leu, or a D-α-amino acid analog thereof; cX 6 is the D-form of Arg, His, Lys, Glu, or Gln, or a D-α-amino acid analog thereof; dX 6 is the D-form of Arg, His, Lys, or Glu, or a D-α-amino acid analog thereof; eX 6 is the D-form of Arg, His, or Lys, or a D-α-amino acid analog thereof; fx 6 is the D-form of Arg, or a D-α-amino acid analog thereof; gX 6 is the D-form of Lys, or a D-α-amino acid analog thereof; or hX 6 is the D-form of His, or a D-α-amino acid analog thereof, or a salt thereof.

[0019] In some embodiments, a D-peptide having an amino acid sequence set forth in either SEQ ID NO: 1 or 2, or a salt thereof, aX 10 is the D-form of Trp, or a D-α-amino acid analog thereof; or bX 10 is the D-form of Tyr, or a D-α-amino acid analog thereof, or a salt thereof.

[0020] In some embodiments, a D-peptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or a salt thereof, aX 11 is the D-form of Trp (W), Tyr (Y), or Gln (Q), or a D-α-amino acid analog thereof; bX 11 is the D-form of Tyr(Y), or cX 11 is the D-form of Trp(W), or a D-α-amino acid analog thereof, or a salt thereof.

[0021] In some embodiments, X 1 From X 6 , X 10 and X 11 and C is the D-form of cysteine, F is the D-form of phenylalanine, and N is the D-form of asparagine.

[0022] In some embodiments, the core TNFα binding domain is [ka] [ka] A D-peptide or a salt thereof is provided, having an amino acid sequence selected from:

[0023] In some embodiments, a D-peptide or a salt thereof is provided, wherein the core TNFα binding domain has amino acids comprising the amino acid sequence set forth in SEQ ID NOs: 77-110.

[0024] In some embodiments, a D-peptide or a salt thereof is provided, further comprising a tag sequence attached to the N-terminus of the peptide. In some embodiments, a D-peptide or a salt thereof is provided, wherein the tag comprises the amino acid sequence D-Asp or D-Asp D-Asp (DD). In some embodiments, a D-peptide or a salt thereof is provided, further comprising a tag sequence attached to the C-terminus of the peptide. In some embodiments, a D-peptide or a salt thereof is 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, a D-peptide or a salt thereof is provided, wherein the N-terminus of the peptide comprises a cap. In some embodiments, a D-peptide or a salt thereof is provided, wherein the cap comprises an acetyl group or a protecting group. In some embodiments, a D-peptide or a salt thereof is provided, wherein the C-terminus of the peptide comprises a cap. In some embodiments, a D-peptide or a salt thereof is provided, wherein the cap comprises an amide group or a protecting group.

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

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

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

[0029] In some embodiments, the trifunctional crosslinker is selected from the group consisting of tris(succinimidyl)aminotriacetate (TSAT), tris-succinimidyl(6-aminocaproyl)aminotriacetate (LC-TSAT), Fmoc scaffold (bis(2,5-dioxopyrrolidin-1-yl)4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-((2,5-dioxopyrrolidin-1-yl) Fmoc scaffold with PEG27 chain, cyclohexa scaffold (tris(2,5-dioxopyrrolidin-1-yl)cyclohexane-1,3,5-tricarboxylate), nitro scaffold (bis(2,5-dioxopyrrolidin-1-yl)4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)heptanedioate), )-4-nitroheptanedioate), an amine scaffold (bis(2,5-dioxopyrrolidin-1-yl)4-amino-4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)heptanedioate), an amine-PEG27 scaffold, a cholesterol scaffold, a heterotetrameric PEG scaffold based on a 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propionic acid scaffold, a multimeric scaffold based on 4-amino-4-(2-carboxyethyl)heptanedioic acid, or 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propionic acid, or a salt thereof.

[0030] In some embodiments, the multimer comprises: 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-cyclohexa; 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 or a salt thereof, wherein the peptide is a D-peptide.

[0031] In some embodiments, [ka] [ka] The present invention provides a D-peptide or a salt thereof, having an amino acid sequence selected from at least one of:

[0032] In some embodiments, [ka] or a salt thereof.

[0033] In some embodiments, pharmaceutical compositions are provided 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, such pharmaceutical compositions are provided that are formulated for parenteral administration. In some embodiments, such pharmaceutical compositions are provided that are formulated for intravenous, intramuscular, or subcutaneous administration. In some embodiments, such pharmaceutical compositions are provided that are formulated for oral administration. In some embodiments, such pharmaceutical compositions are provided that are formulated for topical administration. In some embodiments, such pharmaceutical compositions are provided that are formulated for topical administration to the skin (transdermal) or eye. In some embodiments, such pharmaceutical compositions are provided that are formulated for rectal administration.

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

[0035] In some embodiments, a method of treating a TNFα-mediated disease is provided, comprising administering an effective amount of any of the D-peptides described herein, or a salt or pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a salt thereof, 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 type), hidradenitis suppurativa, uveitis (intermediate uveitis, posterior uveitis, or panuveitis), or non-radiographic axial spondyloarthritis.

[0036] In some embodiments, the TNFα-mediated disease is 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, any of the D-peptides described herein, or a pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a pharmaceutically acceptable salt thereof, or any of the pharmaceutical compositions described herein is administered orally. In some embodiments, any of the D-peptides described herein, or a pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a pharmaceutically acceptable salt thereof, or any of the pharmaceutical compositions described herein is administered rectally. In some embodiments, any of the D-peptides described herein, or a pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a pharmaceutically acceptable salt thereof, or any of the pharmaceutical compositions described herein is administered parenterally.

[0037] In some embodiments, the TNFα-mediated disease is an inflammatory skin disease. In some embodiments, the inflammatory skin disease is selected from psoriasis vulgaris and cutaneous lupus. In some embodiments, the administration of any of the D-peptides described herein, or a pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a pharmaceutically acceptable salt thereof, or any of the pharmaceutical compositions described herein is topical. In some embodiments, the administration of any of the D-peptides described herein, or a pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a pharmaceutically acceptable salt thereof, or any of the pharmaceutical compositions described herein is parenteral. In some embodiments, the administration of any of the D-peptides described herein, or a pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a pharmaceutically acceptable salt thereof, or any of the pharmaceutical compositions described herein is oral.

[0038] In some embodiments, the TNFα-mediated disease is an inflammatory disease such as, for example, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, idiopathic arthritis (polyarticular), or axial spondyloarthritis not meeting radiological criteria. In some embodiments, administration of any of the D-peptides described herein, or a pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a pharmaceutically acceptable salt thereof, or any of the pharmaceutical compositions described herein is parenteral.

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

[0040] In some embodiments, a method for reducing TNFα-mediated inflammation is provided, comprising administering to a subject any of the D-peptides described herein, or a pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a pharmaceutically acceptable salt thereof, or any of the pharmaceutical compositions described herein. In some embodiments, a method for inhibiting TNFα is provided, comprising administering to a subject any of the D-peptides described herein, or a pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a pharmaceutically acceptable salt thereof, or any of the pharmaceutical compositions described herein. In some embodiments, a method for reducing an inflammatory response mediated by TNFα is provided, comprising administering to a subject any of the D-peptides described herein, or a pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a pharmaceutically acceptable salt thereof, or any of the pharmaceutical compositions described herein. In some embodiments, administration is by oral administration, parenteral administration, topical (transdermal) administration, or rectal administration. In some embodiments, any of the D-peptides described herein, or a pharmaceutically acceptable salt thereof, or any of the multimers described herein, or a pharmaceutically acceptable salt thereof, or any of the pharmaceutical compositions described herein, is administered locally to reduce TNFα activity or inflammation or an 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 type), hidradenitis suppurativa, uveitis (intermediate uveitis, posterior uveitis, or panuveitis), or axial spondyloarthritis that does not meet radiographic criteria.

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

[0042] In some embodiments, the peptides described herein, any multimer of D-peptides described herein, or pharmaceutically acceptable salts thereof are provided for use in a method for treating 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 type), hidradenitis suppurativa, uveitis (intermediate uveitis, posterior uveitis, or panuveitis), or axial spondyloarthritis that does not meet radiographic criteria.

[0043] These and other aspects of the present invention can be more fully understood by reference to the following detailed description, non-limiting examples of specific embodiments, and the accompanying drawings.

[0044] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0045] [Figure 1] Figure 1 shows a comparison of TNFα binding by selected peptides isolated by mirror image phage screening from all libraries.

[0046] [Figure 2] FIG. 2 shows a comparison of the binding of peptide 18 and two peptide 18 variants to TNFα in a solid-phase binding assay.

[0047] [Figure 3] FIG. 3 shows that peptide 18 can block the binding of TNFα to its receptor, TNFR1, in a solid-phase binding assay.

[0048] [Figure 4A] FIG. 4A shows that peptide 18 can block the cellular activity of TNFα in an L-929 cell killing assay.

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

[0050] [Figure 5] FIG. 5 shows the effect of amino acid changes at the N-terminus, C-terminus and internal positions of peptide 18 on TNFα binding.

[0051] [Figure 6] FIG. 6 shows the effect of amino acid changes at the N-terminus, C-terminus, and internal positions of peptide 18 using a TNFα / TNFR blocking assay.

[0052] [Figure 7] FIG. 7 shows the effect of amino acid changes at the N-terminus, C-terminus, and internal positions of peptide 18 using a TNFα / TNFR blocking assay.

[0053] [Figure 8] FIG. 8 shows the effect of amino acid alterations at the N-terminus, C-terminus and internal positions of peptide 18 using an L-929 cell killing assay.

[0054] [Figure 9] FIG. 9 shows the effect of inserting an N-terminal PEG4 spacer and inserting or deleting amino acid residues into peptide 18 using a TNFα binding assay.

[0055] [Figure 10] FIG. 10 shows the effect of inserting an N-terminal PEG spacer and inserting or deleting amino acid residues into peptide 18 using an L-929 cell killing assay.

[0056] [Figure 11] FIG. 11 compares the activity of peptide 18 KtoR dimer and trimer using the L-929 cell killing assay.

[0057] [Figure 12] Figure 12 compares the activity of peptide 18 variants and other library hits using a TNFα binding assay.

[0058] [Figure 13] FIG. 13 compares the activity of peptide 18 KtoR trimers with different PEG lengths using the L-929 cell killing assay.

[0059] [Figure 14] FIG. 14 compares the activity of peptide 18 and peptide 18 variants using the L-929 cell killing assay.

[0060] [Figure 15A-1] FIG. 15A shows the structures of the three multimeric scaffolds used to generate peptide trimers. [Figure 15A-2] FIG. 15A shows the structures of the three multimeric scaffolds used to generate peptide trimers. [Figure 15A-3] FIG. 15A shows the structures of the three multimeric scaffolds used to generate peptide trimers.

[0061] [Figure 15B] FIG. 15B compares the activity of the three peptide 18 KtoR trimers using the L-929 cell killing assay.

[0062] [Figure 16A-1] FIG. 16A shows the structures of the six multimeric scaffolds used to generate peptide trimers. [Figure 16A-2]FIG. 16A shows the structures of the six multimeric scaffolds used to generate peptide trimers. [Figure 16A-3] FIG. 16A shows the structures of the six multimeric scaffolds used to generate peptide trimers.

[0063] [Figure 16B] FIG. 16B compares the activity of six peptide 18 KtoR trimers using the L-929 cell killing assay.

[0064] [Figure 17] FIG. 17 compares the activity of peptide 18 KtoR trimers with different PEG lengths using an L-929 cell killing assay.

[0065] [Figure 18] Figure 18 compares the activity of peptide 18 variants using the L-929 cell killing assay.

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

[0067] [Figure 20] FIG. 20 shows the activity of peptide 18 variants containing non-canonical amino acids using the L-929 cell killing assay.

[0068] [Figure 21-1] FIG. 21 shows that fluorescently labeled peptide DD-018 binds to native TNFα. [Figure 21-2] FIG. 21 shows that fluorescently labeled peptide DD-018 binds to native TNFα. [Figure 21-3] FIG. 21 shows that fluorescently labeled peptide DD-018 binds to native TNFα.

[0069] [Figure 22] Figures 22A and B compare the activity of peptide TF-018-KtoR-WW and its oligomers, and an anti-TNFα antibody using a TNFα / TNFR blocking assay.

[0070] [Figure 23] Figures 23A and B compare the activity of peptide TF-018-KtoR-WW and oligomers, and anti-TNFα antibodies using the L-929 cell killing assay.

[0071] [Figure 24] FIG. 24 shows that the peptide TF-18-KtoR-WW C-trimer is able to block the activity of membrane-bound TNFα.

[0072] [Figure 25] FIG. 25 shows the inhibition of IL-8 levels by peptide TF-18-KtoR-WW C-trimer after stimulation of blood samples with recombinant human TNF, LPS and anti-CD3+anti-CD28 antibodies.

[0073] [Figure 26] FIG. 26 shows the results of administration of peptide TF-18-KtoR-WW C-trimer in a human TNFα mouse challenge model.

[0074] [Figure 27] Figures 27 and 28 show the plasma levels of peptide TF-18-KtoR-WW C-trimer after a single subcutaneous dose in CD1 mice. [Figure 28] Figures 27 and 28 show the plasma levels of peptide TF-18-KtoR-WW C-trimer after a single subcutaneous dose in CD1 mice.

[0075] [Figure 29] FIG. 29 shows peptide TF-18-KtoR-WW C-trimer levels in plasma after administration of 5.0 mg / kg IV or 50 mg / kg SC.

[0076] [Figure 30] FIG. 30 shows peptide TF-18-KtoR-WW C-trimer levels in the small intestine, liver, plasma, large intestine and kidney after oral administration.

[0077] [Figure 31] FIG. 31 shows the inhibition of IL-6 after subcutaneous administration of peptide TF-18-KtoR-WW C-trimer to Tg1278 mice.

[0078] [Figure 32] FIG. 32 shows the inhibition of mouse KC after subcutaneous administration of peptide TF-18-KtoR-WW C-trimer to Tg1278 mice. DETAILED DESCRIPTION OF THE INVENTION

[0079] definition For convenience, certain terms in the specification, examples, and claims are defined here. Unless stated otherwise or implicitly from context, the following terms and phrases have the meanings provided below. Since the scope of the present invention is limited only by the claims, definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0080] As used herein, and unless otherwise indicated, the terms "a" and "an" shall be taken to mean "one," "at least one," or "one or more." Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0081] Unless the context clearly requires otherwise, throughout this description and the claims, the words "comprise," "comprising," and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, in other words, "including but not limited to."

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

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

[0084] As used herein, the terms "protein" and "polypeptide" are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues, respectively. The terms "protein" and "polypeptide" also refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, the usage of these terms in the art overlaps. 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 referred to as TNF alpha or TNF-alpha or TNF) is a protein expressed in various 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, those having the amino acid sequences shown 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 α-amino acid residue having the same absolute configuration as D-glyceraldehyde.

[0087] As used herein, amino acids are named herein using either their one-letter or three-letter codes, in accordance with recommendations from IUPAC. Unless otherwise indicated by context, amino acids are of the D-form.

[0088] The phrases "D-forms of standard L-amino acids" and "D-forms of any of the standard L-amino acids" refer to the 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 achiral.

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

[0090] As used herein, "specifically binds" means -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 Specific binding refers to the ability of a binder (e.g., a D-peptide described herein) to bind to a target with a KD of M or lower. Specific binding can be affected, for example, by the affinity and avidity of the binder and the concentration of the target polypeptide. Those skilled in the art can use any suitable method, such as titrating the binder in a suitable cell binding assay or a suitable solid-phase binding assay, to determine appropriate conditions under which a binder selectively binds to a target. A binder that specifically binds to a target cannot be displaced by a dissimilar competitor. In certain embodiments, a binder is said to specifically bind to a target if it preferentially recognizes that target in a complex mixture of proteins and / or macromolecules.

[0091] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristics of that embodiment.

[0092] The term "consisting of" refers to the compositions, methods, and individual components thereof described herein, excluding any element not recited in the description of that embodiment.

[0093] Except as otherwise indicated in the examples, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as being modified in all instances by the term "about." When used in conjunction with percentages, the term "about" can mean + / - 1%.

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

[0095] The term "pharmaceutically acceptable salts" generally refers to salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable. "Pharmaceutically acceptable salts" also includes solvates of such salts, particularly hydrates.

[0096] Other terms are defined herein within the description of various aspects of the invention.

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

[0098] Also provided are methods of using TNFi peptides for the treatment of TNFα-mediated diseases, including inflammatory and autoimmune diseases and disorders. As used herein, the term "TNFα-mediated disease" refers to a disease or disorder in which the TNFα signaling pathway and / or the cell biological effects of TNFα contribute to the disease or its symptoms. In some embodiments, the TNFi peptides are administered parenterally, intravenously, intramuscularly, subcutaneously, orally, topically, or rectally. These and other embodiments are further described herein.

[0099] peptide

[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. TNFi peptides contain a core TNFα-binding domain composed of D-amino acids.

[0101] In some embodiments, there is provided a D-peptide or salt thereof, wherein the peptide comprises a core TNFα binding domain of D-amino acids 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), and X 2 From X 11 each of which is a D-amino acid; 2 is the D-form of any of the common L-amino acids other than 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), Gln (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G); X 5 is the D-form of any of the common L-amino acids other than cysteine; X 6 is the D-form of any of the common L-amino acids other than cysteine; X 7 is the D form of Phe(F); X 8 is the D-type of Asn(N), and X 9 is the D-type of Asn(N), and X 10 is the D-form of Trp (W) or Tyr (Y); X 11is the D-form of Trp (W), Gln (Q), Tyr, or His (H); and C represents the D-form of cysteine, thereby providing a D-peptide or a salt thereof.

[0102] In some embodiments, the peptide comprises a core TNFα binding domain of D-amino acids and has the following amino acid sequence: * -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), and X 2 From X 11 each of which is a D-amino acid; 2 is the D-form of any of the common L-amino acids other than cysteine; X 3 are the D forms of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), and Leu (L); X 4 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 is the D-form of any of the common L-amino acids other than cysteine; X 6 is the D-form of any of the common L-amino acids other than cysteine; X 7 is the D form of Phe(F); X 8 is the D-type of Asn(N), and X 9 is the D-type of Asn(N), and X 10 is the D-form of Trp (W) and Tyr (Y); X 11 is the D-form of Trp (W), Tyr (Y), Gln (Q) or His (H); C represents the D-form of cysteine; * represents an optional intramolecular bond, or a salt thereof.

[0103] In some embodiments, the peptide comprises a core TNFα binding domain of D-amino acids and has the following amino acid sequence: * -X 2 -[W / F / Y]-[Polarity]-X 5 -X 6 -FNN-[W / Y]-WC * (SEQ ID NO: 113), and X 2 is the D form of any of the standard L-amino acids other than Cys, and X 5 is the D-form of any of the standard L-amino acids other than cysteine, and X 6 represents the D-amino acid, which is the D-form of any of the standard L-amino acids other than cysteine, and polarity includes one of R, K, H, E, D, Q, N, T, S, P, A, or G; * provides D-peptides or salts thereof, which optionally exhibit an 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: CX 2 -X 3 -X 4 -X 5 -X 6 -FNNX 10 -X 11 -C (SEQ ID NO: 1), and X 1 From X 6 , X 10 , and X 11 each of which is a D-amino acid; 2 is the D-form of Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), Ile (I), Met (M), or Trp (W), or a D-α-amino acid analog thereof; X 3 is the D-form of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), or Leu (L), or a D-α-amino acid analog thereof; X 4is the D-form of 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-α-amino acid analog thereof; X 5 is the D-form of Pro (P), Trp (W), His (H), Gln (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), or a D-α-amino acid analog thereof; X 6 is the D-form of Arg (R), His (H), Lys (K), Glu (E), Gln (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-α-amino acid analog thereof; X 10 is the D-form of Trp (W) or Tyr (Y), or a D-α-amino acid analog thereof; X 11 is the D-form of Trp (W), Tyr (Y), Gln (Q) or His (H), or a D-α-amino acid analogue thereof; C represents the D-form of cysteine.

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

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

[0107] In some embodiments of the TNFi peptides set forth in SEQ ID NO: 1 or 2, X3 is the D-form of Trp, Phe, Tyr, or Ser. 3 is the D-form of Trp, Phe, or Tyr. 3 is the D form of Trp or Phe. 3 is the D-form of Trp. 3 is the D form of Phe.

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

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

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

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

[0112] In some embodiments of the TNFi peptides set forth in SEQ ID NO: 1 or 2, X 11 is the D-form of Trp (W), Tyr (Y), or Gln (Q). 11 is the D-form of 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: * -X 2 -[W / F / Y]-X 4 -X 5 -X 6 -FNN-[W / Y]-WC * (SEQ ID NO: 3), and X 2 is the D form of Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), Ile (I), Met (M), or Trp (W); X 4is the D-form of 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 Pro(P), Trp(W), His(H), Gln(Q), Arg(R), Ala(A), Val(V), Leu(L), or Gly(G), and X 6 is the D-form of Arg (R), His (H), Lys (K), Glu (E), Gln (Q), Val (V), Leu (L), Ser (S), or Ala (A); * 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 the D-form of Thr, Val, His, Leu, Gln, Ala, Ile, Met, or Trp. 2 is the D-form of Thr, Val, His, Leu, or Gln. 2 is the D form of Thr, Val, His, or Leu. 2 is the D-form of Thr, Val, or His. 2 is the D-form of Thr or Val. 2 is the D-form of Thr. 2 is the D-type of Val.

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

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

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

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

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

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

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

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

[0151] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTFRWRFNNYWC (SEQ ID NO: 85), optionally including 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 including an intramolecular disulfide bond.

[0153] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTFQLRFNNYWC (SEQ ID NO: 87), optionally including an intramolecular disulfide bond.

[0154] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTFQVRFNNYWC (SEQ ID NO: 88), optionally including an intramolecular disulfide bond.

[0155] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CIWQPKFNNYWC (SEQ ID NO: 89), optionally including an intramolecular disulfide bond.

[0156] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTFQRRFNNYWC (SEQ ID NO: 90), optionally including an intramolecular disulfide bond.

[0157] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTFQWSFNNYWC (SEQ ID NO: 91), optionally including an intramolecular disulfide bond.

[0158] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CVFQRHFNNWWC (SEQ ID NO: 92), optionally including 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 including an intramolecular disulfide bond.

[0160] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CAFQHHFNNWWC (SEQ ID NO: 94), optionally including 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 including 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 including an intramolecular disulfide bond.

[0163] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CHFNRRFNNWWC (SEQ ID NO: 97), optionally including 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 including an intramolecular disulfide bond.

[0165] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CLYQLVFNNWWC (SEQ ID NO: 99), optionally including 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 including an intramolecular disulfide bond.

[0167] In some embodiments, the TNFi peptide comprises a core TNFα binding domain having the following amino acid sequence: CTLQQQFNNYWC (SEQ ID NO: 101), optionally including 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 including 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 including 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 including 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 including 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 including 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 including 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 including 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 including 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 including an intramolecular disulfide bond.

[0177] In some embodiments, the TNFi peptide comprises a core TNFα binding domain selected from the following amino acid sequences: 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), in each case optionally comprising an intramolecular disulfide bond.

[0178] In some embodiments, the TNFi peptide comprises a core TNFα binding domain selected from 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-α-amino acid analog of the D-amino acid. A D-α-amino acid analog is a D-α-amino acid analog that has both an amine functional group and a carboxylic acid functional group as either NH, NHR, or NR. In some embodiments, a TNFi peptide having a core TNFα binding domain set forth in any of SEQ ID NOS: 1-3 and 111-113 has at least one D-α-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 of SEQ ID NOS: 1-3 and 111-113 has at least two D-α-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 of SEQ ID NOS: 1-3 and 111-113 has at least three D-α-amino acid analog substitutions in the core TNFα binding domain.

[0181] Charged D-α-amino acid analogs include the D-forms of 4-methylglutamate, mono-4-fluoroglutamate, 4,4-difluoro-glutamate, gamma-hydroxyglutamate, L-threo-β-hydroxyaspartate, epsilon-N,N,N-trimethyllysine, epsilon-N-acetyllysine, aza-leucine, O-phosphoserine, 3-methylhistidine, 5-hydroxylysine, and methylarginine.

[0182] Polar uncharged D-amino acid analogs include the D forms of L-Glu gamma-hydrazide, L-albidiine, L-theanine, beta-hydroxynorvaline, aspartic acid methyl ester, and glutamic acid methyl ester.

[0183] Aromatic D-amino acid analogs include the D-forms of β-2-thiazolyl-alanine, triazolealanine, 3-fluoro-L-tyrosine, 3-nitro-L-tyrosine, 3-fluorophenylalanine, 2-thienylalanine, β-methylphenylalanine, β-thienylserine, N-acetylserine, N-formylmethionine, p-azidophenylalanine, p-ethynylphenylalanine, p-nitro-phenylalanine, 7-azatryptophan, 5-hydroxytryptophan, 5-fluorotryptophan, 5-methoxytryptophan, 3-(thianaphthen-3-yl)-L-alanine, 2-thienylglycine, and L-phenylglycine.

[0184] Nonpolar D-amino acid analogs include the D-forms of 2-aminohex-5-ynoic acid, norleucine, norvaline, ethionine, β-azidohomoalanine, trifluoronorleucine, trifluoronorvaline, LC-propargylglycine, L-allylglycine, β-cyclopropylalanine, 3-fluoro-valine, methyl ether L-threonine, methyl ether L-allo-threonine, 4-thia-isoleucine, L-cyclohexyl-glycine, 5',5',5'-trifluoroleucine, β-cyclopentylalanine, thiazolidine-2-carboxylic acid, thiazolidine-4-carboxylic acid, pseudoproline, 3,4-dehydroproline, and 4-hydroxyproline.

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

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

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

[0188] In some embodiments, the TNFi peptide comprises a core TNFα-binding domain having the following amino acid sequence: CXFQPRFNNWWC (SEQ ID NO: 75), optionally containing an intramolecular disulfide bond, with a D-α-amino acid analog, where X is norleucine (nor-Leu).

[0189] Other modifications to 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, e.g., with an acetyl group. In some embodiments, the TNFi peptide is capped at the N-terminus, e.g., with 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, e.g., with an amide group. In some embodiments, the TNFi peptide is capped at the C-terminus, e.g., with a protecting group.

[0191] In some embodiments, a 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, a 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 tag may, for example, increase the solubility of the TNFi peptide (e.g., in aqueous solution). When more than one tag is present, the tags may 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 both the N-terminus and the C-terminus of the core TNFα-binding domain.

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

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

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

[0195] In some embodiments, TNFi peptides may contain additional amino acids in addition to the core TNFα-binding domain and any attached tags. Such additional amino acids may be D-amino acids or L-amino acids. In some embodiments, TNFi peptides containing tags at the N- and / or C-terminus are 12-50 amino acids in length, or 12-40 amino acids in length, or 12-30 amino acids in length, or 12-20 amino acids in length. In some embodiments, TNFi peptides containing tags at the N- and / or C-terminus are 12-50 amino acids in length. In some embodiments, TNFi peptides containing tags at the N- and / or C-terminus are 12-40 amino acids in length. In some embodiments, TNFi peptides containing tags at the N- and / or C-terminus are 12-30 amino acids in length. In some embodiments, TNFi peptides containing tags at the N- and / or C-terminus are 12-20 amino acids in length.

[0196] Linker

[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 an L-amino acid. In some embodiments, the linker comprises a D-amino acid. In some embodiments, the linker comprises a chemical group other than an amino acid. In some embodiments, the linker comprises 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 an L-amino acid. In some embodiments, the linker comprises a chemical group (other than an amino acid) and a D-amino acid.

[0198] In some embodiments, the linker comprises repeating polymer units. In some embodiments, the polymer units are attached to the N-terminus of the TNFi peptide. In some embodiments, the polymer units are attached to the C-terminus of the TNFi peptide. In some embodiments, the polymer units are attached to both the N-terminus and the C-terminus of the TNFi peptide. The polymer units attached to the N-terminus and the C-terminus may 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 polysarcosine chain. In some embodiments, the polymer unit comprises an alkyl chain. In some embodiments, the polymer unit comprises a polypeptide chain, e.g., albumin, or other polypeptide.

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

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

[0202] In some embodiments, the linker comprises a functional group at the end opposite its attachment site to the TNFi peptide. The functional group can serve as a cap and / or provide an attachment site for another molecule, such as a multimeric scaffold group or an auxiliary 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 an o-pentafluorophenyl ester. In some embodiments, the functional group is one or more amino acid residues. In some embodiments, 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 an attachment site for the 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 may be in the form of multimers, such as dimers or trimers, or higher multimers. For example, if the multimer is a dimer, the dimer may be composed of two identical TNFi peptides (i.e., homodimeric), or two different TNFi peptides (i.e., heterodimeric). The multimer may also be a trimer. If the multimer is a trimer, the trimer may be composed of two identical and one different TNFi peptides (i.e., heterotrimeric), or three identical TNFi peptides (i.e., homotrimeric), or three different TNFi peptides, each of which is distinct from the other (i.e., heterotrimeric).

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

[0206] In some embodiments, the TNFi peptides form dimers. In some embodiments, the TNFi peptides form trimers. In some embodiments, the TNFi peptides form multimers comprising at least four TNFi peptides. In some embodiments, higher order multimers may comprise 5, 6, 7, 8, 9, 10, 11, 12, or more TNFi peptides. Multimers may be heteromeric or homomeric.

[0207] In some embodiments, the TNFi peptides are joined in a dimeric format to form a linear chain. In some embodiments, the TNFi peptides are linked via the C-terminus of one peptide joined to the N-terminus of a second peptide, optionally including a linker connecting the two TNFi peptides. In some embodiments, the TNFi peptides are linked via the C-terminus of one peptide joined to the C-terminus of a second peptide, optionally including a linker connecting the two TNFi peptides. In some embodiments, the TNFi peptides are linked via the N-terminus of one peptide joined to the N-terminus of a second peptide, optionally including a linker connecting the two TNFi peptides. In any of these embodiments, the TNFi peptides may be the same or different.

[0208] In some embodiments, the TNFi peptides are joined in a trimer format to form a linear chain. In some embodiments, the TNFi peptides are linked via the C-terminus of one peptide joined to the N-terminus of the next peptide, and its C-terminus is connected to the N-terminus of a third peptide, optionally including a linker connecting the pair of TNFi peptides. In some embodiments, the TNFi peptides are joined in any suitable orientation of the N- and C-termini. In any of these embodiments, the TNFi peptides may be the same or different.

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

[0210] In some embodiments, TNFi peptides are cross-linked to form multimers, e.g., branched multimers. TNFi peptides are cross-linked via their N- and / or C-termini. In some embodiments, the cross-linking agent is a polyethylene glycol (PEG) group derivatized with a reactive group, e.g., an N-hydroxysuccinimide (NHS)-ester (which reacts with Lys residues) and / or a maleimide group (which reacts with thiol groups). In some embodiments, the cross-linking agent may contain two distinct linking chemistries (e.g., an NHS ester at one end and a maleimide at the other end). In any of these embodiments, the peptides may be the same or different.

[0211] In some embodiments, TNFi peptides are crosslinked using a multimeric scaffold. The multimeric scaffold may contain two or more functional groups for attachment of TNFi peptides, with each TNFi peptide optionally being attached to the multimeric scaffold via a linker. In some embodiments, each TNFi peptide is attached to a reactive group on the multimeric scaffold via a linker, e.g., a PEG group. In some embodiments, each TNFi peptide is attached to a reactive group on the multimeric scaffold via a linker, e.g., a PEG group.

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

[0213] In some embodiments, the orthogonal group can be a functional group for attachment of 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 repeats, or 2 to 24 ethylene glycol repeats. In some embodiments, the orthogonal group is a cholesteryl-PEG4-NHS ester, as shown in the diagram below, where n=4. [ka]

[0214] In some embodiments, the TNFi peptide is attached to the multimeric scaffold via a linker. In some embodiments, the linker comprises a PEG group having a functional group at its terminus. In some embodiments, the PEG group has an NHS ester at each of its two termini. In some embodiments, the PEG group has an o-pentafluorophenyl ester at each of its two termini. In some embodiments, the PEG group has an amine group at one terminus and an NHS ester at the other terminus. In some embodiments, the PEG group has an amine group at one terminus and an o-pentafluorophenyl ester at the other terminus. In some embodiments, the PEG group has a carboxyl group at one terminus and an o-pentafluorophenyl ester at the other terminus. In some embodiments, the PEG group has an amine or carboxyl group at one terminus and an NHS ester or o-pentafluorophenyl ester at the other terminus. In some embodiments, the PEG group has an amine or carboxyl group at one terminus and a carboxyl or amine group at the other terminus.

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

[0216] In some embodiments, the multimeric scaffold is an 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 an Fmoc scaffold with PEG27 chains (see Figure 15A). In some embodiments, the multimeric scaffold is a cyclohexanone scaffold. 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 FIG. 16A. In some embodiments, the multimeric scaffold is a cholesterol scaffold as shown in FIG. 16A. In some embodiments, the heterotetrameric PEG scaffold is a 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)-methyl]propoxy}propionic acid scaffold. In some embodiments, the multimeric scaffold is based on 4-amino-4-(2-carboxyethyl)heptanedioic acid. In some embodiments, the TNFi peptide comprises a C-terminal D-lysine residue attached to the carboxyl group of 4-amino-4-(2-carboxyethyl)heptanedioic acid. In some embodiments, the TNFi peptide multimer comprises three TNFi peptides, each having a C-terminal D-lysine residue attached to the carboxyl group of 4-amino-4-(2-carboxyethyl)heptanedioic acid.In some embodiments, such TNFi peptide multimers further comprise an auxiliary molecule attached to a free amino or carboxyl group of the 4-amino-4-(2-carboxyethyl)heptanedioic acid scaffold. In some further embodiments, the auxiliary molecule is a PEG group.

[0217] In some embodiments, the linker PEG group attached to the multimeric scaffold has 1-48 subunits. In some embodiments, the linker PEG group has 1-30 subunits. In some embodiments, the linker PEG group has 1-24 subunits. In some embodiments, the linker PEG group has 1-12 subunits. In some embodiments, the linker PEG group has 4-30 subunits. In some embodiments, the linker PEG group has 4-24 subunits. In some embodiments, the linker PEG group has 4-12 subunits. In some embodiments, the linker PEG group has 4-10 subunits. In some embodiments, the linker PEG group has 4 subunits. In some embodiments, the linker PEG group has 6 subunits. In some embodiments, the linker PEG group has 8 subunits. In some embodiments, the linker PEG group has 10 subunits. In some embodiments, the linker PEG group has 12 subunits. In some embodiments, the PEG groups of the linker as 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 a first PEG chain and a second PEG chain that are connected in series.In some embodiments, the PEG group can contain an internal NHS ester or other bond, for example, formed by connecting multiple PEG groups together.

[0218] In some embodiments, the linker or multimeric scaffold may include a tris, di-lysine, benzene ring, phosphate, or peptide core as a functional or reactive group. In some embodiments, the crosslinking group includes a bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl glutarate (DSG), or bis(sulfosuccinimidyl)suberate (DST) group. In some embodiments, the crosslinking group includes a thiol-reactive group, such as haloacetyl (e.g., iodoacetate), pyridyl disulfide (e.g., HPDP), and other thiols.

[0219] Ancillary molecules attached to TNFi peptides or their multimers

[0220] In some embodiments, a TNFi peptide or multimer thereof may be modified or linked to auxiliary molecules, such as potency-enhancing molecules, stabilizing molecules, or other molecules that provide increased activity, potency, binding, pharmacokinetics, membrane localization, or other properties.

[0221] In some embodiments, the multimeric scaffold can be coupled to an auxiliary molecule, such as a PEG molecule (e.g., linear or branched), a sterol (e.g., cholesterol) or an 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, keyhole limpet hemacyanin, sperm whale ovalbumin, green fluorescent protein, a gold particle, a magnetic particle, an agarose bead, a lactose bead, an alkane chain (e.g., C8, C1-C6, or C), or a hydroxyl group (e.g., C8, C1-C6, or C8). 1~ C8 alkyl chains, etc.), or fatty acids (e.g., C8 fatty acids, C 16 fatty acids, C 18 In other embodiments, the auxiliary molecule may be a linkage of multiple multimers, e.g., a linkage of multiple trimers (e.g., to increase molecular weight and reduce renal filtration).

[0222] In some embodiments, a 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 attached at 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, e.g., with a biotin group. In some embodiments, the TNFi peptide is labeled at the C-terminus. In some embodiments, the TNFi peptide is labeled at the C-terminus, e.g., with a biotin group.

[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 an orthogonal arm of the multimeric scaffold. In some embodiments, the label or other detectable marker is a biotin group.

[0224] In some embodiments, the auxiliary molecule is attached to the TNFi peptide or multimer thereof via a PEG group. A variety of chemistries known in the art can be used to attach the auxiliary molecule to the PEG group. For example, the auxiliary molecule may be attached to the PEG group via a carbamate formed by a formic acid halide molecule reacting with an amine. In another example, the auxiliary molecule may be attached to the PEG group via an amide bond formed by condensation between a carboxylic acid molecule and an amine. In another example, the auxiliary molecule may be attached to the PEG group via an amide bond formed by an NHS molecule or any other activated ester. In another example, the auxiliary molecule may be attached to the PEG chain via an amide bond formed by the reaction of a ketone with an amine (isourea). In another example, the auxiliary molecule may be attached to the PEG chain via a thioether bond formed by the reaction of a thiol with a maleimide ester. In another example, the auxiliary molecule may be attached to the PEG group via an ether bond, for example, via a dehydration reaction between the terminal hydroxyl on the auxiliary molecule and the PEG group. In yet another example, an auxiliary molecule may be attached to a PEG group via click chemistry, for example, Huisgen 1,3-dipolar cycloaddition between an azide and an alkyne.

[0225] In some embodiments, the PEG group attached to the auxiliary molecule may consist of a single PEG chain or a first and second PEG chain linked in series. In some embodiments, the PEG group on the auxiliary molecule may contain an internal NHS ester or other bond, for example, formed by linking multiple PEG groups together.

[0226] Multimer avidity

[0227] A multimer of TNFi peptides may have increased affinity for TNFα molecules compared to the affinity of a single TNFi peptide or a control peptide for TNFα molecules. In some embodiments, a multimer of TNFi peptides may have increased affinity for trimeric TNFα compared to the affinity of a single TNFi peptide or a control peptide for trimeric TNFα. In some embodiments, a TNFi peptide multimer is a dimer. In some embodiments, a TNFi peptide multimer is a trimer. The single peptide or control peptide may be identical to one of the components of the multimer, or the single peptide may be a different peptide that is not included in the multimer.

[0228] TNFi peptide multimers may exhibit approximately a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or 10,000-fold increase in affinity for trimeric TNFα when compared to the affinity of the TNFi peptide alone.

[0229] Pharmaceutical Composition

[0230] TNFi peptides and multimers thereof can be administered in vivo in pharmaceutical compositions. Pharmaceutical compositions typically contain pharmaceutically acceptable additives, carriers, and / or other components. "Pharmaceutically acceptable" means that the components of the composition are not biologically or otherwise undesirable, i.e., they can be administered to a subject (e.g., a human) together with a TNFi peptide or multimer thereof without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which they are contained. Pharmaceutically acceptable additives are selected to minimize any degradation of the active ingredient (e.g., a TNFi peptide or multimer thereof) and to minimize any adverse side effects in the subject, as is well known to those skilled in the art.

[0231] The pharmaceutical composition may be formulated for any suitable administration form. The pharmaceutical composition may be formulated for oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, or subcutaneous administration), local administration (e.g., transdermal administration), rectal administration, etc. The required dosage of the pharmaceutical composition varies from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the disease, the mode of administration, etc. The appropriate dosage can be determined by one skilled in the art using only routine experimentation, taking into account the teachings of this specification.

[0232] Parenteral administration of pharmaceutical compositions is generally characterized by injection.Injectables can be prepared in any conventional form, such as liquid solution or suspension, solid form suitable for liquid solution or suspension before injection, or emulsion.Parenteral administration can also include the use of slow release or sustained release system (i.e., depot) so that a constant dosage is maintained.

[0233] Suitable additives and carriers, as well as their formulations, are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render 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 may be about 5 to about 8, or about 7 to about 7.5. Additional carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing a TNFi peptide or multimer thereof, which matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and concentration of the administered composition.

[0234] The carrier for inclusion in pharmaceutical compositions is known to those skilled in the art.The most typical of these is the standard carrier for administering drugs to humans, including sterile water, saline and physiological pH buffer solution etc.Composition can be administered intramuscularly or subcutaneously.

[0235] In addition to the TNFi peptide or multimer thereof, the pharmaceutical composition may include a carrier, a thickener, a diluent, a buffer, a preservative, a surfactant, etc. The pharmaceutical composition may also include one or more other active ingredients, such as an antibacterial agent, an anti-inflammatory agent, an anesthetic agent, etc.

[0236] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are 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, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, inert gases, and the like.

[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 may be desirable.In addition, it is contemplated herein that compositions designed for oral administration may further comprise a gut permeabilizing agent.

[0238] In some embodiments, TNFi peptides or multimers thereof may be administered as pharmaceutically acceptable acid or base addition salts 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 ethanolamines.

[0239] In some embodiments, the pharmaceutical composition of a TNFi peptide or multimer thereof is lyophilized. The formulation of a TNFi peptide or multimer thereof can be lyophilized for reconstitution or can be in liquid form. The terms "lyophilization," "lyophilized," and "freeze-dried" refer to a process in which the material to be dried is first frozen, and then the ice or frozen solvent is removed by sublimation under a vacuum. Additives may be included in the formulation before lyophilization to enhance the stability of the lyophilized product during storage. A "reconstituted" formulation is prepared by dissolving the lyophilized protein formulation in a diluent so that the protein disperses in the reconstituted formulation. The reconstituted formulation is suitable for administration (e.g., parenteral administration), and may be suitable for subcutaneous or other routes of administration, as appropriate.

[0240] use

[0241] 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 TNFα activity in a subject. In some embodiments, a TNFi peptide or multimer thereof is administered to reduce an inflammatory response mediated by TNFα. A TNFi peptide or multimer thereof can be administered orally, parenterally, intravenously, intramuscularly, subcutaneously, topically, rectally, etc. In some embodiments, a TNFi peptide or multimer thereof is administered locally to achieve local inhibition of TNFα activity or TNFα-mediated inflammation or a TNFα-mediated inflammatory response.

[0242] TNFi peptides and multimers thereof can be used to treat subjects with TNFα-mediated diseases. In some embodiments, TNFi peptides or multimers thereof are administered to subjects in need thereof for the treatment of TNFα-mediated diseases. In some embodiments, the TNFα-mediated disease is an inflammatory disease or condition. In some embodiments, the TNFα-mediated disease is an autoimmune disorder.

[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] As used herein, the term "subject" refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a livestock animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, for example, a human. In some embodiments, the subject is a human, a monkey, or a dog. In some embodiments, the subject is a human.

[0245] In some embodiments, a TNFi peptide or multimer thereof is administered to a subject in need thereof for the treatment of a TNFα-mediated disease, such as 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 uveitis, posterior uveitis, or panuveitis), or axial spondyloarthritis that does not meet radiographic criteria.

[0246] In some embodiments, the TNFi peptide or multimer thereof is administered to a subject in need thereof who has an inflammatory bowel disease, such as 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, a 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 to the subject rectally (e.g., by enema).

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

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

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

[0251] Effective dosages and schedules for administering TNFi peptides or multimers thereof can be determined empirically, and making such determinations is within the skill of the art. The dosage range for administering TNFi peptides or multimers thereof and compositions is high enough to produce the desired effect in which the symptoms / disorders are affected. The dosage is typically not so high as to cause serious adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, etc. Generally, dosages vary depending on the subject's age, condition, sex, and extent of disease, the route of administration, or whether other drugs are included in the regimen, and can be determined by one of ordinary skill in the art. Dosages can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one or multiple doses per day for one or several days.

[0252] In some embodiments, a typical daily dosage of a TNFi peptide or multimer thereof used may range from about 1 μg to 100 mg / kg of body weight per day or more, depending on the factors listed above. In some embodiments, a typical daily dosage of a TNFi peptide or multimer thereof used may range from about 0.01 μg to 1 mg / kg of body weight per day or more. In some embodiments, a TNFi peptide or multimer thereof may be administered several times a day, daily, weekly, monthly, or yearly, depending on the subject's condition, other treatment modalities, etc. One of ordinary skill in the art can readily ascertain an appropriate dosing schedule.

[0253] The TNFi peptide or multimer thereof may be administered prophylactically to a patient or subject at risk for a TNFα-mediated disease or at risk for a recurrence of a TNFα-mediated disease.

[0254] Methods for producing TNFi peptides and multimers thereof

[0255] TNFi peptides or multimers thereof can be prepared by any method known to those skilled in the art for preparing D-peptides or multimers thereof.TNFi peptides can be linked, for example, by disulfide bridges.For example, the D-peptides disclosed herein have two Cys residues connected by a disulfide bond, which cyclizes the peptide and creates a more compact and structured peptide.This disulfide is known to enhance TNFα binding activity.

[0256] Two or more TNFi peptides can also be linked together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either FMOC (fluorenylmethyloxycarbonyl) or Boc (tertbutyloxycarbonyl) chemistry (Applied Biosystems, Inc., Foster City, CA). One of skill in the art can readily recognize that peptides corresponding to any of the disclosed TNFi peptides can be synthesized, for example, by standard chemical reactions. For example, a TNFi peptide can be synthesized and not cleaved from its synthesis resin, while another fragment of the peptide can be synthesized and then cleaved from the resin, thereby exposing a functionally blocked terminal group on the other fragment. Through peptide condensation reactions, these two fragments can be covalently joined via peptide bonds at their carboxyl and amino termini, respectively, to form TNFi peptides (Grant GA (1992) Synthetic Peptides: A User Guide. WH Freeman and Co., NY (1992); Bodansky M. and Trost B., Ed. (1993) Principles of Peptide Synthesis. SpringerVerlag Inc., NY, both of which are incorporated herein by reference for their material regarding peptide synthesis). Once isolated, these independent peptides can be linked to form peptides through similar peptide condensation reactions.

[0257] For example, enzymatic ligation of cloned or synthetic peptide segments allows for the joining of relatively short peptide fragments to generate larger peptides (Abrahmsen L., et al., Biochemistry, 30:4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be used to synthetically construct larger peptides from shorter peptide fragments. This method consists of a two-step chemical reaction (Dawson et al., Synthesis of Proteins by Native Chemical Ligation. Science, 266:776779 (1994)). The first step is the 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 covalently linked product. Without modification of reaction conditions, this intermediate spontaneously undergoes a rapid intramolecular reaction 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 are chemically linked, where the bond formed between the peptide segments as a result of chemical ligation is a non-natural (non-peptide) bond (Schnolzer, M. et al., Science, 256:221 (1992)). Using this technique, analogs of protein domains, as well as large quantities of relatively pure proteins with full biological activity, have been synthesized (deLisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).

[0259] This invention 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 following amino acid sequence: CX 2 -X 3 -X 4 -X 5 -X 6 -FNNX 10 -X 11 -C (SEQ ID NO: 1), and X 1 From X 6 , X 10 and X 11 are each a D-amino acid or a Dα-amino acid analog thereof, and C, F, and N are the D-forms of cysteine, phenylalanine, and asparagine, or D-α-amino acid analogs thereof; aX 2 is the D-form of Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), Ile (I), Met (M), or Trp (W), or a D-α-amino acid analog thereof; bX 3 is selected from the D-form of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H) or Leu (L), or a D-α-amino acid analog thereof; cX 4is a polar amino acid selected from the D-form of 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-α-amino acid analog thereof; dX 5 is the D-form of Pro (P), Trp (W), His (H), Gln (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), or a D-α-amino acid analog thereof; eX 6 is the D-form of Arg (R), His (H), Lys (K), Glu (E), Gln (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-α-amino acid analog thereof; fx 10 is the D-form of Trp (W) or Tyr (Y), or a D-α-amino acid analog thereof; gX 11 is the D-form of Trp (W), Tyr (Y), Gln (Q) or His (H), or a D-α-amino acid analog thereof; hC represents the D-form of cysteine ​​or its D-α-amino acid analogue, F represents the D-form of phenylalanine or its D-α-amino acid analogue; and N represents the D-form of asparagine or its D-α-amino acid analogue, a D-peptide or a salt thereof. 2. The core TNFα binding domain has the following amino acid sequence: CX 2 -[W / F / Y]-X 4 -X 5 -X 6 2. The D-peptide of embodiment 1, having the formula: -FNN-[W / Y]-WC (SEQ ID NO: 3), or a salt thereof. 3. A D-peptide or a salt thereof according to any one of embodiments 1 to 2, further comprising an intramolecular disulfide bond between cysteine ​​residues in the core TNFα-binding domain. 4.aX 2 is the D-form of Thr, Val, His, Leu, Gln, Ala, Ile, Met, or Trp, or a D-α-amino acid analog thereof; bX 2 is the D-form of Thr, Val, His, Leu, or Gln, or a D-α-amino acid analog thereof; cX 2 is selected from the D-forms of Thr, Val, His, and Leu, or their D-α-amino acid analogs; dX 2 is selected from the D-forms of Thr, Val, and His, or D-α-amino acid analogs thereof; eX 2 is selected from the D-forms of Thr and Val, or D-α-amino acid analogs thereof; fx 2 is the D-form of Thr, or a D-α-amino acid analog thereof; or gX 2 4. The D-peptide or salt thereof according to any one of embodiments 1 to 3, wherein is the D-form of Val or a D-α-amino acid analogue thereof. 5.aX 3 is selected from the D-forms of Trp, Phe, Tyr, and Ser, or D-α-amino acid analogs thereof; bX 3 is selected from the D-forms of Trp, Phe, and Tyr, or D-α-amino acid analogs thereof; cX 2 is selected from the D-forms of Trp and Phe, or D-α-amino acid analogs thereof; dX 3 is the D-form of Trp, or a D-α-amino acid analog thereof; or eX 3 is the D-form of Phe, or a D-α-amino acid analog thereof. 6.aX 4 is the D-form of Arg, His, Gln, Asn, Lys, Thr, or Ser, or a D-α-amino acid analog thereof; bX 4 is selected from the D-form of Arg, His, Gln, and Asn, or their D-α-amino acid analogs; cX4 is the D-form of Arg, His, or Gln, or a D-α-amino acid analog thereof; dX 4 is the D-form of Arg, Gln, or Asn, or a D-α-amino acid analog thereof; or eX 4 is the D-form of Gln, or a D-α-amino acid analog thereof. 7.aX 5 is the D-form of Pro, Trp, His, Gln, Gly, Arg, Ala, Val, or Leu, or a D-α-amino acid analog thereof; bX 5 is the D-form of Pro, Trp, His, Gln, Gly, Arg, or Val, or a D-α-amino acid analog thereof; cX 5 is the D-form of Pro, Trp, or His, or a D-α-amino acid analog thereof; dX 5 is the D-form of Pro or Trp, or a D-α-amino acid analog thereof; or eX 5 7. The D-peptide or salt thereof according to any one of embodiments 1 to 6, wherein is the D-form of Pro, or a D-α-amino acid analogue thereof. 8.aX 6 is the D-form of Arg, His, Lys, Glu, Gln, Val, Leu, Ser, or Ala, or a D-α-amino acid analog thereof; bX 6 is the D-form of Arg, His, Lys, Glu, Gln, Val, or Leu, or a D-α-amino acid analog thereof; cX 6 is the D-form of Arg, His, Lys, Glu, or Gln, or a D-α-amino acid analog thereof; dX 6 is the D-form of Arg, His, Lys, or Glu, or a D-α-amino acid analog thereof; eX 6is the D-form of Arg or His, or a D-α-amino acid analog thereof; fx 6 is the D-form of Arg, or a D-α-amino acid analog thereof; or gX 5 8. The D-peptide or salt thereof according to any one of embodiments 1 to 7, wherein is the D-form of His, or a D-α-amino acid analogue thereof. 9.aX 10 is the D-form of Trp, or a D-α-amino acid analog thereof; or bX 10 is the D-form of Tyr or a D-α-amino acid analog thereof. 10.aX 11 is the D-form of Trp (W), Tyr (Y), or Gln (Q), or a D-α-amino acid analog thereof; or bX 11 is the D-form of Trp(W) or a D-α-amino acid analog thereof. 11.X 1 From X 6 , X 10 , and X 11 11. The D-peptide or salt thereof according to any one of embodiments 1 to 10, wherein each of the following is a D-α-amino acid: 12. The core TNFα binding domain is [ka] [ka] 12. The D-peptide or salt thereof according to any one of embodiments 1 to 11, having the amino acid sequence: 13. The D-peptide or salt thereof of any of the preceding embodiments, wherein the core TNFα binding domain has the amino acid sequence of SEQ ID NOs: 77-110. 14. A D-peptide or salt thereof according to any 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. A D-peptide or salt thereof according to any 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. A D-peptide or salt thereof according to any of the previous 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. A D-peptide or salt thereof according to any of the previous embodiments, wherein the C-terminus of the peptide comprises a cap. 21. A D-peptide or salt thereof according to embodiment 20, wherein the cap comprises an amide group or a protecting group. 22. A D-peptide or salt thereof according to any of the preceding embodiments, further comprising a polyethylene glycol (PEG) group. 23. A D-peptide or salt thereof according to any 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. A D-peptide or salt thereof according to any of embodiments 22 or 24, wherein the PEG group is attached to the N-terminus of the D-peptide. 26. A D-peptide or salt thereof according to any of embodiments 22 or 24, wherein the PEG group is attached to the C-terminus of the D-peptide. 27. A D-peptide or salt thereof according to any of embodiments 22 and 24-26, wherein each PEG group is selected from PEG groups 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 PEG groups having 6 subunits, 8 subunits, 10 subunits or 12 subunits. 29. A multimer of a D-peptide according to any 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 of embodiments 29-31, further comprising a multimeric scaffold attached to a D-peptide, optionally via a linker. 33. The multimer or salt thereof according to embodiment 32, wherein the multimeric scaffold is trimeric. 34. The multimer or salt thereof according to embodiment 32, wherein the multimeric scaffold is tetrameric. 35. Trifunctional crosslinkers include tris(succinimidyl)aminotriacetate (TSAT), tris-succinimidyl(6-aminocaproyl)aminotriacetate (LC-TSAT), and the Fmoc scaffold (bis(2,5-dioxopyrrolidin-1-yl)4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-((2,5-dioxopyrrolidin-1- ... The scaffolds were: Fmoc scaffold (tris(2,5-dioxopyrrolidin-1-yl)cyclohexane-1,3,5-tricarboxylate), nitro scaffold (bis(2,5-dioxopyrrolidin-1-yl)4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)heptanedioate), Fmoc scaffold with PEG27 chain, cyclohexa scaffold (tris(2,5-dioxopyrrolidin-1-yl)cyclohexane-1,3,5-tricarboxylate), nitro scaffold (bis(2,5-dioxopyrrolidin-1-yl)4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)heptanedioate), 35. The multimer or salt thereof of any of embodiments 33 or 34, wherein the multimer is a PEG scaffold based on 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propionic acid scaffold, a PEG scaffold based on 4-amino-4-(2-carboxyethyl)heptanedioic acid, or 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propionic acid. 36. Multimers are 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-cyclohexa; 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 or a pharmaceutically acceptable salt thereof, of embodiment 35, wherein the multimer has a structure selected from: 37. The following: [ka] [ka] 29. 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 the following: 38. The following: [ka] 37. 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 the following: 39. A pharmaceutical composition comprising at least one D-peptide or multimer as set forth 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 of embodiment 39, which is formulated for parenteral administration. 41. The pharmaceutical composition of embodiment 40, which is formulated for intravenous, intramuscular, or subcutaneous administration. 42. The pharmaceutical composition of embodiment 39, which is formulated for oral administration. 43. The pharmaceutical composition of embodiment 39, which is formulated for topical administration. 44. The pharmaceutical composition of embodiment 43, which is formulated for topical administration to the skin (transdermal) or eye. 45. The pharmaceutical composition of 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 a pharmaceutically acceptable salt thereof, and a stabilizer. 47. A lyophilized composition of the pharmaceutical composition according to any one of embodiments 39 to 45 and a stabilizer. 48. A rewetting solution for the freeze-dried composition according to embodiment 46 or 47. 49. A method for treating a TNFα-mediated disease, comprising administering an effective amount of a D-peptide according to any one of embodiments 1 to 28 or 37, a multimer according to any one of embodiments 29 to 36 and 38, or a pharmaceutical composition according to any one of embodiments 39 to 45, or a pharmaceutically acceptable salt thereof. 50. The method of embodiment 49, wherein 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 uveitis, posterior uveitis, or panuveitis), or axial spondyloarthritis not fulfilling radiographic criteria. 51. The method of embodiment 49, wherein the TNFα-mediated disease is inflammatory bowel disease. 52. The method of embodiment 51, wherein the inflammatory bowel disease is adult Crohn's disease, pediatric Crohn's disease, or ulcerative colitis. 53. The method of embodiment 51 or 52, wherein administration is oral. 54. The method of embodiment 51 or 52, wherein administration is rectal. 55. The method of embodiment 51 or 529, wherein administration is parenteral. 56. The method of embodiment 49, wherein the TNFα-mediated disease is an inflammatory skin disease. 57. The method of embodiment 56, wherein the inflammatory skin disease is psoriasis vulgaris, hidradenitis suppurativa, or cutaneous lupus. 58. The method of embodiment 56 or 57, wherein administration is topical. 59. The method of embodiment 56 or 57, wherein administration is parenteral. 60. The method of embodiment 49, wherein the TNFα-mediated disease is an inflammatory disease, and the inflammatory disease is systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or juvenile idiopathic arthritis (polyarticular type). 61. The method of embodiment 60, wherein administration is parenteral. 62. The method of embodiment 65, 59 or 61, wherein the parenteral administration is selected from intravenous, subcutaneous, and intramuscular. 63. A method for reducing TNFα-mediated inflammation, comprising administering to a subject a D-peptide or multimer thereof described in any one of embodiments 1 to 38, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in any one of embodiments 39 to 45. 64. A method for inhibiting TNFα, comprising administering to a subject a D-peptide or multimer thereof described in any one of embodiments 1 to 38, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in any one of embodiments 39 to 45. 65. A method for reducing an inflammatory response mediated by TNFα, comprising administering to a subject a D-peptide or a multimer thereof described in any one of embodiments 1 to 38, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in any one of embodiments 39 to 45. 66. The method of any of embodiments 63-65, wherein administration is by oral administration, parenteral administration, topical (transdermal), or rectal administration. 67. The method of any of embodiments 63-65, wherein the D-peptide or multimer thereof, or a pharmaceutically acceptable salt thereof, is administered locally to reduce TNFα activity or inflammation or an inflammatory response. 68. The method of any of embodiments 63-67, wherein the subject has a TNFα-mediated disease. 69. The method of embodiment 68, wherein 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 uveitis, posterior uveitis, or panuveitis), or axial spondyloarthritis not fulfilling radiographic criteria. 70. A D-peptide or multimer thereof according to any of embodiments 1 to 38, or a pharmaceutically acceptable salt thereof, for use as a medicament. 71. A D-peptide or multimer thereof according to any of embodiments 1-38, or a pharmaceutically acceptable salt thereof, for use in a method of treating a subject by therapy. 72. The D-peptide of embodiment 71, wherein the subject has a TNFα-mediated disease. 73. The D-peptide of embodiment 72, wherein 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 type), hidradenitis suppurativa, uveitis (intermediate uveitis, posterior uveitis, or panuveitis), or axial spondyloarthritis not fulfilling radiographic criteria.

[0260] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Specific embodiments of, and examples thereof, are described herein for illustrative purposes; however, various equivalent modifications are contemplated within the scope of the present disclosure, as will be recognized by those skilled in the relevant art. The teachings of the present disclosure provided herein may be applied to other procedures or methods, where appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, if necessary, to employ the compositions, functions, and concepts of the above references and applications to provide still further embodiments of the present disclosure. These and other changes can be made to the present disclosure in light of the detailed description.

[0261] Particular elements of any of the foregoing embodiments can be combined with or substituted for elements in other embodiments. Furthermore, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages to be within the scope of the present disclosure.

[0262] All identified patents and other publications are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the 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 an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or presentation of the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents. [Example]

[0263] Example

[0264] Example 1 Screening of a phage display library for TNFα antagonist peptides

[0265] To identify D-peptides that bind to and inhibit the activity of human TNFα, a peptide library was screened using mirror-image phage display, an enantiomer screening technique. A synthetic biotinylated D-amino acid form of human TNFα (i.e., a mirror image of the natural L-amino acid form) was used to identify L-amino acid peptides (L-peptides) that specifically bind to the D-amino acid form of TNFα. The sequences of the L-peptides that bound to the D-amino acid form of human TNFα ("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 native human TNFα (L-amino acid form) and inhibit its activity.

[0266] Synthesis of the L-form of TNFα

[0267] A biotinylated version of human TNFα was synthesized and shown to have stability and activity similar to the native TNFα protein. The human TNFα protein has the following amino acid sequence, corresponding to Ref Seq NP_000585.2: [ka] had the following characteristics:

[0268] Human TNFα L-form was synthesized using established solid-phase peptide synthesis-based chemical approaches (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). Each synthetic intermediate was purified to homogeneity by reverse-phase HPLC and characterized by HPLC and LCMS analysis. The final linear protein target was then refolded by first forming a single intramolecular disulfide and then dialyzing stepwise from 6 M to 2 M, 0.5 M, and 0 M guanidine hydrochloride buffer. The folded biotinylated L-TNFα trimer was isolated by SEC (size exclusion chromatography) to remove any aggregated or misfolded species (i.e., separation of trimers from monomers or higher-order aggregates). The synthetic and recombinant L-TNFα proteins were further characterized by biophysical (LC and ESI-MS), biochemical (multiple assays for binding to TNFR), and activity assays (L-929 cell killing assay) to ensure they were comparable (data not shown).

[0269] Synthesis of the D-form of TNFα

[0270] Biotinylated D-TNFα protein for screening in mirror-image phage display was synthesized using D-amino acids. The folded conformation of the synthetic D-form of TNFα protein was confirmed by electrospray-mass spectrometry. Folding was confirmed by a 2 Da difference and a different ionization pattern due to the formation of one disulfide bond. The D-protein was purified by size exclusion chromatography, and this trimeric form was used in the following studies.

[0271] Mirror-image phage display selection process

[0272] Mirror-image phage display was performed on the folded biotinylated D-TNFα trimeric protein by screening a phage library of L-peptides with nine different shapes (one linear and eight disulfide-restricted). 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); an L-peptide library having the sequence X2CX8CX2 (where C is cysteine, each X is any amino acid except cysteine, and the subscript numbers refer to the number of L-amino acids at each position in the peptide); 10 L-peptide libraries having C (where C is cysteine, each X is any L-amino acid other than cysteine, and the number of subscripts refers to the number of L-amino acids between the cysteine ​​residues); and the general formula CX n Six libraries of lariat L-peptides with C (where C is cysteine, each X is any L-amino acid, and n refers to the number of L-amino acids between the cysteine ​​residues (n = 2, 4, 6, 8, 10, 12)) yielded a total unique sequence diversity of approximately 4 x 10 10 It was.

[0273] Both next-generation deep sequencing and Sanger sequencing of phage binding to biotinylated D-TNFα trimeric protein were used to identify "hit" sequences (highest affinity binders). Bioinformatics analysis of the sequences identified >10 distinct consensus sequences (related families of peptides with certain residues in common) (data not shown). Multiple hits from each family were tested in phage ELISA assays to identify the best binders to biotinylated D-TNFα trimeric protein. Twenty-eight hits identified during the selection process were further validated using an assay quantifying the binding of different phage clones to biotinylated D-TNFα protein. Multiple hits from each family, prioritized based on consensus sequence abundance and strength, were synthesized as D-peptides for testing for binding to L-TNFα and blocking L-TNFα binding to its receptor, as described below. Twenty-eight phages identified during the selection process were further validated using an assay to quantify the binding of different phage clones to biotinylated D-TNFα protein. Multiple hits from each family, prioritized based on the abundance and strength of consensus sequences, were synthesized as D-peptides for testing for binding to L-TNFα protein and blocking L-TNFα binding to its receptor, as described below. Peptides identified by sequencing include those with the core TNFα-binding domain shown in the Examples below and in SEQ ID NOs: 78-111.

[0274] Identification of early D-peptide binders to TNFα

[0275] The top 28 hits from 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-TNFα using a sequential solid-phase ELISA binding assay in which the D-peptides were captured on neutravidin-coated plates. Soluble His6-tagged TNFα was detected using an anti-His-HRP secondary antibody. Referring to Figure 1, TNFα-018 emerged as the top hit based on its binding to recombinant TNFα (L form) and its high aqueous solubility. Another hit, TNFα-001, was insoluble in aqueous buffer but showed strong binding to TNFα based on phage display.

[0276] Validation of TNFα-binding by the D-peptide hits was confirmed in three different ELISA formats: "solid phase" (as described above), "solution phase" (all components were mixed in solution and then captured on a neutravidin surface), and "sequential solid phase" (biotinylated D-peptides were mixed with TNFα in solution, captured on a neutravidin surface, and then a secondary Ab was added). Binding to L-TNFα by the D-peptide hits was also confirmed using a direct fluorescent readout assay (immobilized recombinant biotinylated TNFα was treated with fluorescently labeled D-peptides). These same fluorescent peptides were also used to demonstrate TNFα-binding by coelution with recombinant TNFα (L form) on an analytical size-exclusion column (SEC) under native conditions. As described below, select D-peptide point mutants abolished 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, which showed that the interaction of the D-peptide (TNF-018α, specific) with TNFα increased its thermal stability (Tm). In addition, SPR (surface plasmon resonance) demonstrated that immobilized TNFα bound to the nascent TNFα-018 D-peptide.

[0277] Example 2 Binding of peptide 18 to TNFα in vitro

[0278] Peptide 18 (also designated TNFα-018 and peptide 018) and the 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.

[0279] Peptide 18 contained the core TNFα-binding domain CVWQPKFNNYWC (SEQ ID NO: 4) and an N-terminal biotin molecule attached to a D-AspAsp (DD) dipeptide tag attached to the N-terminus of the peptide core TNFα-binding domain. The dipeptide tag increased the peptide's solubility. Peptide 18 also contained a C-terminal amide group. Peptides 18-V1 and 18-V2 contained the same core TNFα-binding domain, acetylated at their N-termini, and contained a C-terminal GGEEEK (SEQ ID NO: 30) or GGRRRK (SEQ ID NO: 31) peptide, respectively, and a biotin-amide group. These C-terminal sequences were included in peptides 18-V1 and 18-V2 to determine whether they increased solubility and / or binding. Peptide 18 variants were prepared that had a similar structure to peptide 18, except for a W to G substitution at the penultimate C-terminal residue of the core TNFα-binding domain. The sequences of the peptides are as follows, with amino acid sequence differences from peptide 18 underlined: [ka]

[0280] In the assay, peptides were immobilized on neutravidin plates and washed with wash buffer (PBS pH 7.4 containing 0.1% BSA and 0.01% Tween®-20). His6-TEV-TNFα was then added, followed by washing. The TNFα used in the assay was recombinantly expressed human TNFα with an N-terminal His6 tag. The amount of TNFα bound to the peptide was then measured using an anti-His6 tag antibody conjugated to horseradish peroxidase (HRP), followed by detection using QuantaBlu™ fluorogenic peroxidase substrate (Thermo Scientific).

[0281] Referring to Figure 2, peptides 18, 18-V1, and 18-V2 demonstrated significant binding to TNFα by this capture assay. For the peptide 18 variant, a Trp (W) to Gly (G) substitution adjacent to the C-terminal Cys residue of the core TNFα-binding domain abolished binding of that peptide to TNFα. The BSA control also demonstrated negligible TNFα binding. In Figure 2, the number above each bar indicates the fold increase in peptide binding to TNFα compared to the BSA control. In this assay, differences in the amount of peptide binding to TNFα depended, in part, on the relative position and composition of the peptide tag and the location of the biotin molecule.

[0282] Example 3 Peptide 18 blocked the binding of TNFα to TNFR1

[0283] The peptide biotin DD-18 (biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32)) was tested in a binding assay measuring disruption of binding between TNFα 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 on a neutravidin plate. In parallel, recombinant TNFα with an N-terminal His6 tag (as described in Example 2) was preincubated with peptide 18 at 10, 25, and 50 micromolar concentrations. After preincubation, the TNFα-peptide 18 mixture was then added to the immobilized TNFR1. BSA protein was included as a negative control. A recombinantly expressed llama anti-TNFα nanobody VHH2 (Beirnaert et al., 2017, Front. Immunol. 8:867) was used as a positive control.

[0284] Referring to Figure 3, peptide 18 showed concentration-dependent blocking of TNFα-TNFR1 binding. BSA protein did not disrupt TNFα-TNFR1 binding, while the positive control anti-TNFα nanobody VHH2 blocked TNFα-TNFR1 binding. In Figure 3, the percentage above each bar indicates the percentage reduction in TNFα binding to TNFR1 compared to the negative control. This study showed that peptide 18 can bind to TNFα and block its binding to TNFR1.

[0285] Example 4 Peptide 18 blocked the cellular activity of TNFα in L-929 cells

[0286] The ability of peptide 18 to block TNFα-mediated cell death was demonstrated using an L-929 cell death assay (Trost and Lemasters, 1994, Anal. Biochem. 220(1):149-153). TNFα in the presence of actinomycin D causes TNFα-TNFR-mediated cell death in this cell line. The sequence of peptide 18 was Ac-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32; peptide DD-18).

[0287] Cell death was measured by absorbance at 450 nm. Medium alone (without TNFα) was used as a negative control. TNFα and BSA (without peptide 18) were used as a positive control.

[0288] Referring to Figure 4A, TNFα induced L-929 cell death (compare the first and second columns from the left). Addition of 50 micromolar peptide 18 blocked TNFα-induced cell death (compare the second and third columns from the left). The reduction in TNFα-induced cell death was comparable to that exhibited by soluble TNFR1 (2.5 micrograms / mL), while anti-TNFα VHH2 nanobody (30 micrograms / mL) and anti-TNFα monoclonal antibody (research-grade adalimumab; R&D Systems) showed greater inhibition of TNFα-induced L-929 cell death.

[0289] The concentration of peptide 18 was also varied in this assay from 50 micromolar to 0.78125 micromolar, and cell killing was measured (by absorbance at 450 nm) as described above. Referring to Figure 4B, the left bar in each triplicate shows the results for the indicated concentration of peptide 18 (designated BDP 18; peptide biotin DD-18; SEQ ID NO: 32) alone, the middle bar shows the results for the addition of the indicated amount of peptide 18 (designated BDP 18; peptide biotin DD-18; SEQ ID NO: 32) at a concentration of 0.5 nM, and the right bar shows the results for the addition of the indicated amount of peptide 18 at a concentration of 0.1 nM (Note: the leftmost bar is absent in triplicates 8-9 because peptide 18 was not present (rTNFα only and VHH2)).

[0290] In general, as the amount of peptide 18 decreased, the amount of TNFα-induced cell death increased. The control TNFα-only condition (rTNFα only) caused cell death, as expected. TNFα and anti-TNFα VHH2 nanobody (VHH2) blocked TNFα-induced cell death. 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 medium-only control (medium) gave the expected results.

[0291] In this study, the EC values ​​for peptide 18 at 0.5 nM TNFα and 0.1 nM TNFα were 50 The values ​​were calculated to be 9.6 micromolar and 6.1 micromolar, respectively.

[0292] Example 5 Comparison of the blocking activity of peptide 18 and other leads in the L-929 cell killing assay

[0293] Additional peptide variants of peptide 18 were screened for inhibition of TNFα-induced cell death 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 variants [ka] SEQ ID NO: 35; peptide biotin DD-18 mutant), and a second mutant, peptide 18 di-mutant [ka] SEQ ID NO: 36; peptide biotin DD-18 dimutant). Differences between peptide 18 and peptide 18 mutant and peptide 18 dimutant are underlined in the peptide 18 mutant and peptide 18 dimutant sequences.

[0294] Figure 5 shows the results. In each pair of bars, the left bar shows the results with the peptide alone. The right bar shows the results with the peptide and 0.5 mM recombinant human TNFα. Compared to peptide 18, neither the peptide 18 mutant nor the two mutants showed any significant inhibition of TNFα-induced cell death.

[0295] A trimerized version of peptide 18, peptide 18-IZ (CVWQPKFNNYWC-PEG12-IKKEIEAIKKEQEAIKKKIEAIEKEA-hydrazide; comprising peptide 18 (SEQ ID NO: 4), PEG12, and an IZ coiled-coil (SEQ ID NO: 46)), was tested in this assay. This peptide induces the naturally occurring trimeric IZ coiled-coil sequence, which forms trimers of peptide 18 in solution. Referring again to Figure 5, trimeric peptide 18-IZ showed less inhibition in this assay than peptide 18 as a monomer.

[0296] Example 6 Modification of peptide 18 with a peptide tag

[0297] In peptide 18 (biotin-DDCVWQPKFNNYWC-amide; SEQ ID NO: 32; peptide biotin DD-18), an N-terminal DD dipeptide tag was added to the core TNFα-binding domain (CVWQPKFNNYWC; SEQ ID NO: 4) to increase the solubility of the peptide. In Example 2, two other variants of peptide 18, peptide 18-V1 (SEQ ID NO: 33) and peptide 18-V2 (SEQ ID NO: 34), with C-terminal tags (GGEEEK (SEQ ID NO: 30) and GGRRRK (SEQ ID NO: 31)), respectively, were tested and shown to retain TNFα-binding activity. To determine whether the tag affected the binding activity of the core TNFα-binding domain to TNFα, peptide 18-V1 (designated peptide 18-E3) was analyzed. [ka] and peptide 18-V2 (designated peptide 18-R3) [ka] 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 biotin DD-18-ex, an extended peptide based on the sequence of a peptide isolated from the phage library (Example 1), was 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). [ka] was tested.

[0298] Referring again to Figure 5, peptide 18, which has an N-terminal DD dipeptide, exhibited greater inhibition of TNFα-induced cell death than peptide 18-E3 and peptide 18-R3. Peptide 18-ex exhibited similar or slightly greater inhibition of TNFα-induced cell death than peptide 18.

[0299] Example 7 Comparison of binding of additional peptide 18 sequence variants in the TNFα binding assay

[0300] Using the TNFα binding assay described above (Example 2), the binding of peptide 18 was compared to additional sequence variants. 10 Another peptide from the C library, hit 16, was tested. The following peptides were tested, with sequence differences from peptide 18 underlined: [ka]

[0301] 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 the left). Peptides from both preparations showed comparable binding activity.

[0302] Comparing peptide 18 (biotin-DD-18; SEQ ID NO: 32) with the DGA N-terminal variant (biotin-DGA-18; SEQ ID NO: 38), peptide 18 showed better binding (first three columns from the left), suggesting that the DD peptide tag is preferred over the DGA peptide tag.

[0303] Comparing peptide 18 (biotin-DD-18; SEQ ID NO: 32) with a peptide 18 variant (biotin-DD-18-KtoR; SEQ ID NO: 39) with a Lys to Arg substitution (KtoR) in the core TNFα binding domain, the Lys to Arg substitution enhanced binding (compare the first two columns from the left with the fourth column).

[0304] Comparing the binding of the peptide biotin-DD-18-KtoR (with a DD N-terminal tag; SEQ ID NO: 39) and the peptide biotin-DD-18-KtoR-R3 (SEQ ID NO: 40), the addition of a C-terminal GGRRRK (SEQ ID NO: 31) appeared to further increase binding. Addition of a second biotin molecule to the peptide biotin-DD-18-KtoR-R3 (SEQ ID NO: 40) to create the peptide biotin2-DD-18-KtoR-R3 (SEQ ID NO: 41) did not appear to significantly affect binding. Binding of hit 16 (SEQ ID NO: 42) was similar to that of peptide 18.

[0305] Example 8 Comparison of additional peptide 18 sequence variants in the TNFα-TNFR blocking assay

[0306] Using the TNFα / TNFR blocking assay described above (Example 3), the blocking ability of peptide 18 was compared to additional sequence variants of peptide 18. Hit 16 from the same library was also tested. The peptides tested are shown below. Sequence differences from peptide 18 are underlined. Additionally, some peptides that were not biotinylated were tested in this study. [ka]

[0307] 7, the percentage above each bar indicates the percentage of peptide-dependent blockade of TNFα-TNFR1 binding. First, two different preparations of peptide 18 (biotin-DD-18) were tested (Example 7), both of which showed similar blocking activity (compare the second and third bars from the left).

[0308] In this blocking assay, both non-biotinylated peptide 18 (DD-18) and biotinylated peptide 18 (biotin-DD-18) showed better blocking than the peptide biotin-DD-18-ex (compare the four left columns). Changing the N-terminal tag of peptide 18 from DD to DGA did not significantly affect blockade of TNFα-TNFR1 binding (compare the second through sixth columns from the left). The presence or absence of biotin in the N-terminal DGA variant did not appear to significantly affect blockade by that peptide.

[0309] When the sequence of the core TNFα binding domain was modified by a Lys to Arg substitution (DD-18-KtoR and biotin-DD-18-KtoR; SEQ ID NO: 39), the blocking capacity of these peptides increased compared to the peptide biotin-DD-18 (compare columns 2 and 3 from the left with columns 11 and 12).

[0310] When a C-terminal tag (GGRRRK (SEQ ID NO: 31)) was added to the core modified peptides with or without an additional biotin molecule (biotin-DD-18-KtoR-R3; SEQ ID NO: 40 and biotin2-DD-18-KtoR-R3; SEQ ID NO: 40), the blocking activity of the peptides was reduced (compare columns 11 to 14 from the left).

[0311] Hit 16 with a DD N-terminal addition (DDCHFNPRFNNWWC-amide (SEQ ID NO: 41)) with or without an additional biotin molecule (DD-Hit 16 and biotin-DD-Hit 16) showed comparable blocking compared to peptide DD-18 (SEQ ID NO: 32) (compare columns 4 and 15-16 from the left).

[0312] In summary, these results suggest that biotin did not contribute to the blocking capacity of the peptide and that Lys to Arg substitutions in the core TNFα binding domain increase binding and blocking by the peptide.

[0313] Example 9 Comparison of 18 additional peptide sequence variants in the L-929 cell killing assay

[0314] The L-929 cell killing assay described above (Example 4) was used to compare the ability of peptide 18 (designated DD-18; SEQ ID NO: 32) and additional sequence variants to block TNFα-induced cell death. Biotinylated and non-biotinylated forms of hit 16 were also included. The peptides tested are listed below. Amino acid sequence differences from peptide 18 are underlined. Some peptides were not biotinylated in this study. [ka] [ka]

[0315] Referring to Figure 8, first, two different preparations of peptide 18 (biotin-DD-18; SEQ ID NO: 32) were tested, and both peptide preparations showed somewhat different but comparable blocking activity (compare the second and third bars from the left). Peptide 18 without the biotin tag (DD-18) blocked cell killing better than peptide 18 with biotin (biotin-DD-18) (compare the second, third, and fourth columns from the left). Similarly, peptide 18 without biotin (DD-18) blocked cell killing better than peptide biotin-DD-18-ex (SEQ ID NO: 37) (compare the first and fourth columns from the left).

[0316] Changing the N-terminal tag of peptide 18 from DD to DGA (biotin-DGA-18 and DGA-18; SEQ ID NO: 38), with or without biotin, reduced the peptide's ability to block cell killing in this assay (compare column 4 with columns 5 and 6), a result consistent with the results of these peptides in the TNFα binding assay and TNFα / TNFR assay (above) in Examples 7 and 8.

[0317] 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) increased the ability of the peptide to block cell death (compare column 4 with columns 7 and 8), a result consistent with the results of these peptides in the TNFα-binding assay and TNFα / TNFR assay (above) in Examples 7 and 8.

[0318] When a C-terminal tag (GGRRRK (SEQ ID NO: 31)) was added to the core modified peptides (biotin-DD-18-KtoR-R3; SEQ ID NO: 40 and biotin2-DD-18-KtoR-R3; SEQ ID NO: 41) with or without an additional biotin molecule, the ability of the peptides to block cell killing was reduced (compare columns 7-10). This result is consistent with the results of these peptides in the TNFα binding assay and TNFα / TNFR assay (above) in Examples 7 and 8.

[0319] Hit 16 with the DD N-terminal addition (DDCHFNPRFNNWWC-amide (SEQ ID NO: 41)) with or without an additional biotin molecule (DD-Hit 16 and Biotin-DD-Hit 16) showed comparable blocking compared to peptide DD-18 and peptide DD-18 (SEQ ID NO: 32) (compare columns 2, 3, and 4 from the left with columns 11 and 12).

[0320] In summary, the results of the studies in Examples 7-9 show that modifying the core TNFα-binding domain by substituting Lys with Arg increased the activity of the peptide. The addition of N- and C-terminal peptide tags modulated the activity of the peptide. Biotinylation had little effect on the activity of the peptide.

[0321] Example 10 EC for peptide 18 and variants 50 Determining Values

[0322] The three most active peptides from the above assay were tested in the L-929 cell killing assay (described above in Example 4) at peptide concentrations ranging from 50 micromolar to 0.78125 micromolar (serial two-fold dilutions), and the EC 50 Values ​​were determined (data not shown). The following peptides were tested: [ka]

[0323] EC for each peptide 50 The values ​​were as follows: DD-18: 16.1 micromolar DD-18 KtoR: 6.16 micromolar Biotin-DD-18 KtoR: 8.7 micromolar

[0324] Example 11 Peptide phage library Lariat CX 10 Results from sequence analysis from C.

[0325] Peptide 18 was isolated from the phage library Lariat CX 10 C(CX2X3X4X5X6X7X8X9X 10 X 11 C). Deep sequence analysis of peptide hits from this library revealed that the most frequent amino acids at each position of the peptides (from left to right between cysteine ​​residues) were as follows: X2T, V, H, L, Q, A, I, M, W, X3F, W, Y, S, H, L X4Q, R, N, S, T X5P, W, H, Q, R, A, V, L X6R, H, K, E, Q, V, L, S, A X7F X8N X9N X 10 W, Y X 11W, Y, Q, H

[0326] Peptides from this library consistently exhibited a nucleotide sequence at peptide positions X7 to X 10 The amino acid sequence of FNN(W / Y) was found to be FNN(W / Y).

[0327] Example 12 Comparison of peptide 18 and variant binding in TNFα binding assays

[0328] Peptide 18 and variants described below with various 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-terminally spaced variants with PEG4 (four PEG subunits) were prepared by insertion of PEG4 at the N-terminus between the biotin and the N-terminus of the DD-core TNFα binding domain of peptide 18. The following peptides were tested: [ka]

[0329] Referring to Figure 9, the addition of a PEG4 spacer at the N-terminus of peptide 18 did not significantly affect the binding of the peptide to TNFα (compare columns 1-3 from the left). In addition, the TNFα binding of two different preparations of the peptide biotin-DD-18-KtoR was tested. Little difference in TNFα binding was detected (compare columns 1-2 from the left).

[0330] Peptide 18 cyclizes by disulfide bond formation between the terminal cysteine ​​residues of the core TNFα-binding domain. To determine whether deletion or insertion of a single amino acid between the cysteine ​​residues could alter the peptide's activity, a valine deletion (Biotin-DD-18-no Val) and an alanine insertion (Biotin-DD-18-plus Ala) were prepared. The sequences are as follows: [ka]

[0331] Referring again to Figure 9, deletion or insertion of amino acid residues adjacent to the conserved N-terminal cysteine ​​residue significantly reduced the binding of the peptide to TNFα (compare column 2 with columns 4 and 5). The peptides biotin-DD-18-no Val (SEQ ID NO: 43) and biotin-DD-18-plus Ala (SEQ ID NO: 44) were also tested in the L-929 cell killing assay and showed little ability to inhibit TNFα-induced cell death (data not shown). This result suggested that the spacing of certain residues in the core TNFα-binding domain and the conformation of the peptide may be important for binding to TNFα.

[0332] Example 13 Comparison of PEG-modified peptide 18 and the trimeric version of peptide 18 in the L-929 cell killing assay

[0333] TNFα can assemble as a trimer in vivo. To determine whether multimerization affected the activity of the peptides, peptide 18 and its variants were tested in the L-929 cell killing assay described in Example 4. First, monomeric peptide 18 (peptide DD-18KtoR; SEQ ID NO: 39), which has a Lys to Arg substitution in the core TNFα binding domain, was tested with and without the addition of N-terminal Lys-PEG12 (12 PEG subunits) or C-terminal PEG12-Lys. The monomeric peptide had the following structure: DD-18KtoR:DDCVWQPRFNNYWC-amide (SEQ ID NO: 39) DD-18-KtoR-PEG12-K:Ac-DDCVWQPRFNNYWC-PEG12-K-amide (SEQ ID NO: 44) K-PEG12-DD-18-KtoR:Ac-K-PEG12-DDCVWQPRFNNYWC-amide (SEQ ID NO: 45)

[0334] Referring to Figure 10, addition of Lys-PEG12 at the N-terminus or PEG12-Lys at the C-terminus did not significantly alter the blockade of TNFα-induced cell killing (compare columns 1 and 8 and 9).

[0335] The activity of peptide 18 (biotin-DD-18) and its N-terminal PEG4 version was also tested. The peptide had the following structure: Biotin-DD-18: Biotin-DDCVWQPKFNNYWC-amide (SEQ ID NO: 32) Biotin-PEG4-DD-18: Biotin-PEG4-DDCVWQPKFNNYWC-amide (SEQ ID NO: 54)

[0336] Referring again to Figure 10, the addition of PEG4 to the N-terminus of monomeric peptide 18 did not significantly alter the blockade of TNFα-induced cell death (compare second and third columns from the left).

[0337] Peptide 18 (DD-18) was trimerized by a method in which peptide DD-18-tricasso was assembled on a solid-phase resin. The trimeric peptide DD-18 trimer (tricasso) was directly assembled using semi-orthogonal protecting groups (Dde and Fmoc) (see WO 2017 / 040350 for a general description of the tricasso trimerization method). Additionally, peptide 18 (DD-18-KtoR; SEQ ID NO: 39) with a Lys to Arg substitution was trimerized to form the peptide DD-18-KtoR-IZ using the naturally occurring trimeric IZ coiled-coil sequence (IKKEIEAIKKEQEAIKKKIEAIEKEA (SEQ ID NO: 46)) and a PEG12 spacer. IZ forms a non-covalent trimer. The resulting peptide structure was as follows: DD-18-tricasso:[DDCVWQPKFNNYWC-PEG12]3-KK-amide DD-18-KtoR-IZ:DDCVWQPRFNNYWC-PEG12-IKKEIEAIKKEQEAIKKK IEAIEKEA-hydrazide (SEQ ID NO: 39 linked to SEQ ID NO: 47 via PEG12)

[0338] 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).

[0339] A valine deletion (no biotin-DD-18-Val) and an alanine insertion (biotin-DD-18-plus Ala) of peptide 18 were also tested in this assay. The sequence is as follows: Without biotin-DD-18-Val: biotin-DDCWQPKFNNYWC-amide (SEQ ID NO: 42) Biotin-DD-18-plus Ala: Biotin-DDCAVWQPKFNNYWC-amide (SEQ ID NO: 43)

[0340] Referring again to Figure 10, deletion or insertion of amino acid residues adjacent to the conserved N-terminal cysteine ​​residue significantly reduced binding of the peptide to TNFα (compare second row from the left with rows four and five).

[0341] EC of the respective trimeric peptides DD-18-tricasso and DD-18-KtoR-IZ in the L-929 cell killing assay 50 The EC values ​​were determined by varying the concentration of the peptide from 50 micromolar to 0.78125 micromolar (serial two-fold dilutions). EC values ​​for the trimeric peptide DD-18-KtoR-IZ in PBS and water were 50 The EC values ​​were determined to be 2.4 micromolar and 2.1 micromolar, respectively. 50 The value was determined to be 3.4 micromolar.

[0342] Example 14 Preparation and testing of covalently linked dimeric and trimeric peptides

[0343] Covalently linked dimers and trimers of the peptide DD-18-KtoR were prepared and tested for activity in an L-929 cell killing assay. The 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 installing a lysine residue and a PEG group at the N- or C-terminus of the peptide. Dimers and trimers were then prepared using NHS-amine chemistry with a linker containing multiple NHS groups. The dimers and trimers were purified using standard HPLC purification methods.

[0344] In this study, two different monomers of DD-18-KtoR were prepared: either an N-terminal or C-terminal lysine was added, separated from the DD-18-KtoR peptide by a PEG spacer (SEQ ID NOs: 45 and 46). To prepare the dimer, the DD-18-KtoR peptide bearing PEG-Lys was reacted with an NHS-PEG-NHS linker. To prepare the trimer, the DD-18-KtoR peptide bearing PEG-Lys was reacted with an Fmoc-amino-tri-NHS linker; in this case, Fmoc was retained on the amine of the trimer linker.

[0345] The peptides tested in the L-929 cell killing assay were: 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

[0346] Referring to Figure 11, both covalently linked dimers and trimers of peptide DD-18-KtoR showed significantly greater inhibition of TNFα-mediated cell killing than the monomeric peptide DD-18-KtoR. The dimers and trimers were also more active than the anti-TNFα VHH2 nanobody control.

[0347] Example 15 EC for dimers and trimers of peptide DD-18-KtoR 50 Determining Values

[0348] EC of DD-18-KtoR dimer and trimer prepared in Example 14 50 To determine the values, concentrations of the following peptides, DD-18-KtoR dimer and trimer (SEQ ID NOs: 45 and 46), are varied from 10 micromolar to 0.0015 micromolar (eight two-fold dilutions) in an L-929 cell killing assay. The peptides tested were: 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

[0349] Calculated EC 50 The values ​​were as follows: DD-18-KtoR-N-dimer: 10nM DD-18-KtoR-C-dimer: 2.6nM DD-18-KtoR-N-trimer: 2nM DD-18-KtoR-C-trimer:<<1.5nM

[0350] For both N-linked and C-linked peptides, trimers were more potent than dimers. Both dimeric and trimeric C-linked peptides were more potent than N-linked peptides.

[0351] Example 16 Determination of species selectivity of DD-18-KtoR trimers

[0352] The C-terminally linked DD-18-KtoR trimer from Example 14 was tested for binding activity against 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 (3-fold dilutions). TNFα from these species can bind to human TNFR, so the L-929 cell killing assay could be used to evaluate the blocking activity of the peptide DD-18-KtoR with different TNFα proteins. The trimer used was DD-18-KtoR-C-trimer:Fmoc-[Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3.

[0353] The C-terminally linked trimer was active against human, monkey, and canine TNFα in the cell killing assay at all concentrations tested (data not shown). The trimer was not active against rat TNFα in this assay at any of the concentrations tested (data not shown).

[0354] Example 17 Identification of residues in the monomeric peptide DD-18 involved in interaction with TNFα

[0355] To identify the residues of peptide DD-18 involved in its interaction with TNFα, additional mutants of peptide DD-18 (peptide 18) were prepared. The peptides were tested in the TNFα binding assay described in Example 2. The peptides tested were the following, with differences from peptide Bio-DD-18 underlined: [ka]

[0356] In addition, the N-terminal DD tag was changed to DDD in the following peptides: [ka]

[0357] Referring to Figure 12, peptides with Trp and Asn to Gly substitutions (at positions 3 and 9 of the core TNFα-binding domain, respectively) significantly reduced binding activity (compare column 11 with columns 1 and 2). Peptide biotin-DD-18K2R (with a Lys to Arg substitution at position 6 of the core TNFα-binding domain; SEQ ID NO: 39) exhibited higher binding activity than the previously shown peptide Bio-DD-18 (SEQ ID NO: 32) (compare columns 10 and 11). Addition of another D-Asp residue at the N-terminus 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).

[0358] In addition, CX 10 Other hits from the C-peptide library were tested in the TNFα binding assay. The peptides tested were the following, with differences from peptide Bio-DD-18 underlined: [ka]

[0359] Referring again to Figure 12, peptides Bio-DD-Hit19, Bio-DD-Hit92, and Bio-DD-Hit193 exhibited reduced but some TNFα binding activity compared to peptide DD-018, while peptide Bio-DD-Hit33 exhibited significant binding activity (compare the 6th, 7th, and 9th bars from the left with the 11th bar).

[0360] To assess the contribution of the proximity of the N-terminal DD tag to the core TNFα-binding domain, a PEG4 molecule was inserted between the DD tag and the N-terminus of the core TNFα-binding domain. The activity of this peptide was compared to a peptide with a PEG4 molecule attached 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)

[0361] Referring again to FIG. 12, separation of the DD tag from the N-terminus of the core TNFα peptide binding domain reduced TNFα binding activity to some extent (compare the 4th, 5th and 11th bars from the left).

[0362] Example 18 Modification of the N-terminal DD tag of the monomeric peptide Bio-DD-18

[0363] To further evaluate the effect of alterations in sequence and the proximity of the N-terminal DD tag to the core TNFα binding domain, the following peptides were tested in the L-929 cell killing assay and showed an EC 50 The 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)

[0364] L-929 cell killing assays were performed as described in Example 4.

[0365] EC 50 The 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

[0366] In view of these results, compared to peptide Bio-DD-018 (SEQ ID NO: 32), the addition of an additional Asp residue in the N-terminal tag (SEQ ID NO: 50) resulted in a more effective (lower) EC 50 While removal of the Asp residue (SEQ ID NO: 57) resulted in a less effective (higher) EC 50 Increasing the spacing between the DD tag and the core TNFα binding domain results in less effective (higher) EC 50 Finally, the peptide Biotin-DD-018 (K2R; SEQ ID NO: 39) has a lower EC value than the peptide Bio-DD-018, as previously shown. 50 had value.

[0367] Example 19 Thermal stability of complexes of TNFα and the monomeric peptide biotin-DD-18 and selected variants

[0368] The thermal stability of complexes of TNFα and the peptide biotin-DD-18 and the following mutants was determined using the high-throughput thermal scanning method described by Lavinder et al., J. Am. Chem. Soc. 2009, 131(11):3794-3795. Briefly, the fluorescent dye SYPRO™ was conjugated to the protein (or protein-peptide) complex in solution. The dye fluoresces after the complex denaturation. The results are determined as TNFα-ΔTm (degrees Celsius).

[0369] The following peptides were complexed with TNFα for this study, with differences from biotin-DD-18 underlined: [ka]

[0370] The results were as follows: Bio-18: 80.5 (+5.7 over TNFα alone) Bio-18-WtoG-1: 75.1 (+0.3 over TNFα alone) Bio-18-WtoG-2: 75.0 (+0.2 over TNFα alone) Bio-18-NtoG: 74.7 (-0.1 lower than TNFα alone) TNFα alone 74.8

[0371] Each of the single amino acid substitutions in biotin-DD-18 reduced the thermal stability of the TNFα / peptide complex, consistent with the previously shown reduced binding activity of these peptides.

[0372] Example 20 Thermal stability of the monomeric peptide biotin-DD-18 and spacer modifications

[0373] The thermal stability of DD spacer modifications in the peptide biotin-DD-18 was determined using the thermal stability assay described in Example 19. The peptides tested were: 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)

[0374] The results were as follows: Biotin-PEG4-DD-018: 80.3 (+5.5 over TNFα alone) Bio-DD-PEG4-018: 78.4 (+3.6 over TNFα alone) TNFα alone: ​​74.8

[0375] These results are consistent with the analysis of these peptides described above and indicate 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.

[0376] Example 21 Thermal stability of monomeric peptides with modified N-terminal DD tags of peptide DD-18

[0377] The thermostability assay described in Example 19 was performed on the following peptides to further evaluate changes in the sequence of the N-terminal DD tag of the 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)

[0378] The results were as follows: DD-018: 80.5 (+5.7 over TNFα alone) D-018: 78.9 (+4.1 over TNFα alone) DDD-018: 81.6 (+6.8 over TNFα alone) TNF: 74.8

[0379] These results are consistent with the analysis of these peptides described above and indicate that increasing the number of Asp residues in the N-terminal tag increases TNFα binding and complex stability.

[0380] Example 22 Thermal stability of peptide Bio-DD-018 and variants

[0381] The thermostability assay described in Example 19 was performed on the following peptides from the library screening described in Example 1 to further evaluate the effect of altering the sequence of the peptide Biotin-DD-18 on binding to TNFα. The following peptides were tested, with differences from the peptide Biotin-DD-18 underlined: [ka]

[0382] The results were as follows (°C): DD-018: 80.5 (+5.7 over TNFα alone) DD-018-KtoR: 81.9 (+8.1 over TNFα alone) DD-Hit 19:76.6 (+1.8 over TNFα alone) DD-Hit 33: 77.5 (+2.7 over TNFα alone) DD-Hit 92: 77.2 (+2.7 over TNFα alone) DD-Hit 193: 75.9 (+1.1 over TNFα alone) DD-Hit 16:79.9 (+5.1 over TNFα alone) TNFα alone: ​​74.8

[0383] Peptides representing other hits from the library showed lower thermal stability than peptides Bio-DD-18 and Bio-DD-018-KtoR, whereas the thermal stability of peptide DD-hit 16 was close to that of peptide DD-018.

[0384] Example 23 Analysis of the effect of changing the length of the PEG spacer in the peptide DD-18-KtoR trimer

[0385] Trimers with different PEG spacers were prepared to determine the effect of PEG spacer length on the activity of peptide DD-18-KtoR (SEQ ID NO: 39) trimers in the L-929 cell killing assay. Peptide DD-018-KtoR-C-Trimer(PEG12) from Example 14 was used. Two different preparations of 018-KtoR-C-Trimer(PEG12) were tested in this study. They are designated -1 and -2. In addition, trimers with PEG spacers of 4 and 8 PEG subunits were prepared. The trimeric peptides tested were: 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

[0386] Referring to Figure 13, the PEG8 and PEG12 spacers were approximately equally active, while the PEG4 spacer consistently gave lower binding activity (compare the 2nd, 3rd, and 4th bars with the 1st bar within each series).

[0387] Example 24 Activity of peptide DD-18-dimer and trimer in cell killing assays using mouse TNF

[0388] In Example 16, we showed that peptide 018-KtoR-C-trimer and peptide 018-KtoR-C-dimer bound to human, monkey, and canine TNFα, but not rat TNFα. In this study, the dimers and trimers were tested against mouse TNFα in an L-929 cell killing assay.

[0389] The peptides tested were: 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

[0390] In this study, neither the dimer nor the trimer exhibited significant activity in binding to mouse TNFα (data not shown).

[0391] Example 25 Modification of peptide DD-18 sequence

[0392] CX 10 Peptide 001 from the C library 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 and peptide 018-KtoR, modified peptide 018 was designed. The sequences of the core TNFα-binding domains of peptide 001, peptide 018-KtoR, and modified peptide 18 are shown below, with differences from peptide 018-KtoR underlined: [ka]

[0393] Another variant of peptide DD-018 was also tested, this variant was prepared with an N to Q substitution near the C-terminus of the peptide (the penultimate C-terminal position of the core TNFα binding domain). These peptides were tested in the TNFα binding assay (Example 2). Another peptide, CX 10 Hit 14 from the C library was tested due to its sequence similarity to peptide DD-018. The peptides used in this study were as follows, with differences from the peptide biotin-DD-018 underlined: [ka]

[0394] Each peptide was tested in this study using two different preparations of human TNFα.

[0395] Referring to Figure 14, assays using each preparation of TNFα are shown for each peptide tested (left and right bars within each pair). The peptide biotin-DD-018-KtoR showed slightly better TNFα binding than the peptide biotin-DD-018, which is consistent with previous results. The peptide biotin-DD-018-NtoQ showed almost no TNFα binding activity compared to the peptide biotin-018-KtoR. This result suggests that residues at the C-terminus of the peptide are involved in contact with TNFα. The modified peptide biotin-DD-TF-018-KtoR had slightly better TNFα binding than the peptide biotin-DD-018-KtoR. Hit 14 showed somewhat lower binding than the peptide biotin-DD-018.

[0396] Example 26 EC of modified DD-018-KtoR peptide using L-929 cell killing assay 50 Determining Values

[0397] To further analyze the effect of modifications to the peptide biotin-DD-018, the following peptides were tested in the L-929 cell killing assay to demonstrate an EC 50 Determine the values ​​and underline the differences from peptide biotin-DD-018: [ka]

[0398] EC 50 The values ​​were: 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-Hit 14: 14.7 micromolar

[0399] These results indicated that the TF substitution proximal to the N-terminal cysteine ​​residue in peptide Bio-DD-TF-018-KtoR improved the activity of peptide Bio-DD-018 KtoR. In addition, the addition of an additional aspartic acid residue to the N-terminal tag improved the EC of the peptide. 50 Hit 14 had an EC value similar to that of the peptide biotin-DD-018. 50 The values ​​were shown.

[0400] Example 27 Analysis of further modifications of peptide Bio-DD-018-KtoR

[0401] Based on the results of the previous example, additional peptides containing TF substitutions or variants thereof were designed. These peptides were tested in the L-929 cell killing assay to determine their EC 50 The peptides tested were the following, with differences from peptide Bio-DD-018-KtoR underlined: [ka]

[0402] The decided EC 50 The values ​​were as follows: Bio-DD-018-KtoR: 2.25 micromolar Bio-DD-TF-018-KtoR: 1.16 micromolar 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

[0403] These results also showed that the TF and F substitutions at the N-terminus of the core TNFα-binding domain increased its activity. Both Tyr and Trp at the second C-terminal position of the core TNFα-binding domain allow for comparable binding activity.

[0404] Example 28 Activity of peptide DD-TF-018-KtoR trimer compared to anti-TNFα antibody and VHH2 in L-929 cell killing assay

[0405] The activity of the trimeric form of DD-TF-018-KtoR (SEQ ID NO: 59), which has a PEG8-K spacer attached to it in each arm, was compared to the activity of other TNFα blockers in the L-929 cell killing assay, and the EC 50 The values ​​were determined. Anti-TNFα antibodies were from R&D Systems (research grade). Anti-TNFα VHH2 nanobodies were as described in Beirnaert et al., 2017, Front. Immunol. 8:867. The peptides tested were: DD-TF-018-KtoR C-trimer(PEG8):Fmoc-[Ac-DDCTFQPRFNNYWC-PEG8-K-amide]3

[0406] EC50 The values ​​were as follows: DD-TF-018-KtoR C-trimer (PEG8):1pM~1fM Anti-TNFα antibody: 90.5pM Anti-TNFα VHH2 nanobody: 16.9 pM

[0407] In this assay, the peptide DD-TF-018-KtoR trimer was more potent than the anti-TNFα antibody and the anti-TNFα VHH2.

[0408] Example 29 Analysis of variation in trimeric scaffold structure and spacer length in the L-929 cell killing assay

[0409] To determine whether and / or how different trimer scaffolds affect the activity of peptide DD-018-KtoR (SEQ ID NO: 32), three different scaffolds were tested. In addition, peptide DD-TF-018-KtoR (SEQ ID NO: 58) was included in one trimer construct. Referring to Figure 15A, the scaffolds tested were Fmoc-scaffold (Quanta BioDesign), Fmoc-PEG27 scaffold (Quanta BioDesign), and cyclohexa scaffold (cyclohexatri-NHS ester was prepared from cyclohexanetricarboxylic acid, also known as 1,3,5-cyclohexanetricarboxylic acid, by conversion of the tricarboxylic acid compound with NHS (N-hydroxysuccinimide) in the presence of a carbodiimide (such as DIC or DCC)). The Fmoc-scaffold was previously used in Example 14. In addition, the length of the PEG spacer was varied from PEG8 to PEG6. The trimeric peptides were tested in the L-929 cell killing assay using peptide concentrations from 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 O18-KtoR-trimer(PEG12)-cyclohexa: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-cyclohexa

[0410] Referring to Figure 15B, the choice of scaffold did not appear to significantly affect the activity of the trimers, although trimers with a PEG6 spacer had reduced activity compared to the PEG8 spacer previously tested (see Example 23).

[0411] Example 30 Further analysis of variations in trimeric scaffold structure in L-929 cell killing assays

[0412] To determine whether other trimeric scaffolds increased the activity of the trimeric peptide DD-018-KtoR (SEQ ID NO: 39), five different scaffolds were tested in addition to the Fmoc-PEG27 scaffold used in Example 29. Referring to Figure 16A, the scaffolds tested were a nitro-scaffold (prepared from 4-(2-carboxyethyl)-4-nitroheptanedioic acid by conversion of the tricarboxylic acid compound with NHS (N-hydroxysuccinimide) in the presence of a carbodiimide (such as DIC or DCC)), an amine-PEG27-scaffold (Quanta BioDesign), an Fmoc-scaffold (see Example 29), an amine-scaffold (Quanta BioDesign), a cholesterol-scaffold (Quanta BioDesign), and a biotin-PEG27-scaffold (Quanta BioDesign). The trimeric peptides were tested in the L-929 cell killing assay using peptide concentrations from 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 O18-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 O18-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 O18-KtoR-C-trimer(PEG12)-PEG27-cholesterol: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-PEG27-cholesterol

[0413] Referring to FIG. 16B, in general, the choice of trimeric scaffold did not significantly affect the activity of the trimeric peptides, although the cholesterol-scaffold produced slightly lower activity at lower concentrations.

[0414] Example 31 Activity of certain trimeric peptides at lower concentrations

[0415] Trimers of peptide DD-018-KtoR (SEQ ID NO: 39) or peptide TF-DD-018-KtoR (SEQ ID NO: 59) using the Fmoc-scaffold, amine-scaffold or cyclohexa-scaffold from Examples 29 and 30 were tested in the L-929 cell killing assay at lower concentrations than in the previous examples, and showed EC 50 The trimeric peptide concentrations ranged from 0.32 nM to 0.16 fM (5-fold dilutions). The trimeric peptides tested were: 018-KtoR-C-trimer(PEG12)-Fmoc: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-Fmoc O18-KtoR-C-Trimer(PEG12)-amine: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-amine O18-KtoR-C-Trimer(PEG12)-Cyclohexa: [Ac-DDCVWQPRFNNYWC-PEG12-K-amide]3-cyclohexa TF-018-KtoR-C-trimer(PEG12)-Fmoc: [Ac-DDCTFQPRFNNYWC-PEG12-K-amide]3-Fmoc

[0416] EC 50 The values ​​were as follows: 018-KtoR-C-trimer(PEG12)-Fmoc:5.1pM 018-KtoR-C-trimer(PEG12)-amine: 0.16 fM O18-KtoR-C-trimer(PEG12)-cyclohexa: 4 pM TF-018-KtoR-C-trimer(PEG12)-Fmoc:0.16fM

[0417] O18-KtoR-C-trimer(PEG12)-amine and TF-018-KtoR-C-trimer(PEG12)-Fmoc produced comparable activity. In the Fmoc-trimer format, TF-018-KtoR trimer produced better activity than O18-KtoR trimer. The amine trimer produced the best activity for O18-KtoR trimer.

[0418] Example 32 Analysis of further modifications to the monomeric DD-VF-018-KtoR-WW peptide in the L-929 cell killing assay

[0419] Based on the above examples, 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, peptide VF-018-KtoR-WW (DDCVFQPRFNNWWC; SEQ ID NO: 64), additional substitutions were made in the peptide and tested in the L-929 cell killing assay. The peptides tested are as follows, with differences from peptide VF-018-KtoR-WW (SEQ ID NO: 65) underlined: Peptide Bio-018-KtoR (SEQ ID NO: 39) was also included as a control. [ka] [ka]

[0420] 17, changing the N-terminal aspartic acid in the tag to glutamic acid did not significantly alter the activity of peptide Bio-VF-018-KtoR-WW (SEQ ID NO: 65). Peptides VF-018-QtoKbio-KtoR-WW and Bio VF-018-QtoKbio-KtoR-WW (SEQ ID NO: 67) (with a substitution of Gln (at position 4 (N-terminus) of the core TNFα-binding domain) to Lys or Lys-biotin) showed some reduction in activity compared to Bio-VF-018-KtoR-WW (SEQ ID NO: 65).

[0421] Example 33 Analysis of further modifications to the monomeric DD-VF-018-KtoR-WW peptide in the L-929 cell killing assay

[0422] To determine whether further modifications to peptide VF-018-KtoR-WW (SEQ ID NO: 65) would improve its activity, additional substitutions were made and tested in the L-929 cell killing assay. The peptides tested are as follows, with differences from 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)

[0423] Referring to 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. When the concentration was reduced, peptides Bio-VF-018-KtoR-WW and Bio-VF-018-PtoH-KtoR-WW retained better activity than the other peptides, but all peptides retained binding activity that was comparable to that of peptide Bio-018-KtoR.

[0424] Example 34 Analysis of further modifications to the monomeric and dimeric forms of peptide DD-018-KtoR in the L-929 cell killing assay

[0425] The DD tag at the N-terminus of the peptide provides a negative charge at neutral pH, which helps improve the peptide's solubility. Two aspartic acid (D) residues in the N-terminal tag maintained high solubility and good activity. In this study, the N-terminal Asp (D) was replaced with a mimetic suc (succinate) 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. Both the monomeric and dimeric forms of the peptide DD-018-KtoR-PEG8-K were tested in an L-929 cell killing assay. The dimeric form was produced using amine-NHS chemistry and an NHS-PEG8-NHS crosslinker (Quanta BioDesign). The peptide Bio-DD-TF-018-KtoR-WW (SEQ ID NO: 62) was also included. The peptides tested were: 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)

[0426] Referring to Figure 19, replacement of one Asp residue of the tag with a succinate group did not significantly affect the activity of the monomer and dimer compared to the unmodified peptide.

[0427] Example 35 Analysis of further modifications to peptide TF-018-KtoR-WW in the L-929 cell killing assay

[0428] In this study, D-α-amino acid analogs were site-selectively introduced to determine whether they increased the potency of the monomeric peptide TF-018-KtoR-WW (SEQ ID NO: 62) in an L-929 cell killing assay. The D-α-amino acid analogs used were the methylcysteine ​​analog penicillamine (Pen); the leucine (Leu) analog homoleucine (HomoLeu); and the leucine (Leu) analog norleucine (NorLeu). The peptides tested were the following, with differences in peptide Bio-DD-TF-018-KtoR-WW (SEQ ID NO: 62) underlined: [ka]

[0429] Referring to Figure 20, substitution with D-α-amino acid analogs did not significantly affect the potency of the monomer at the highest concentration, although the peptide Bio-HomoLeu-F-018-KtoR-WW showed somewhat lower activity compared to the other peptides.

[0430] Example 36 Thermal stability of complexes of TNFα with peptide DD-018-KtoR (SEQ ID NO: 39) and oligomeric variants

[0431] The thermal stability assay described in Example 19 was performed on the following TNFα and peptide complexes to further evaluate the effect of oligomerization on TNFα binding stability. The following peptides were tested: DD-18-KtoR: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

[0432] In addition, TNFα alone was included as a reference.

[0433] The results were as follows (°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) TNFα alone 74.8

[0434] These results indicate that increasing the oligomerization of peptide DD-018-KtoR increased the thermal stability of the peptide complex with TNFα.

[0435] Example 37 Fluorescently labeled peptide DD-018 was used to confirm binding of the monomeric peptide to native TNFα (trimeric).

[0436] Native TNFα (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 (by amide coupling of carboxyfluorescein during solid-phase peptide synthesis (SPPS)). The mixture of peptide and TNFα was then assayed by analytical SEC in 0.1 M ammonium acetate, pH 8, using an AKTA analytical SEC column. 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)

[0437] Referring to Figure 21, for each panel, the left y-axis is absorbance at 280 nm, while the right y-axis is absorbance at 480 nm. P1 identifies the elution peak corresponding to TNFα. P2 identifies the elution peak for the free peptide. The top panel shows the elution profile for TNFα alone. The middle panel shows the elution profile for TNFα and fluorescently labeled peptide DD-018. Some peptide co-elutes with TNFα (P1), indicating that it bound to TNFα. P2 is excess peptide. The bottom panel shows the elution profile for TNFα and the non-binding control, fluorescently labeled peptide DD-018-WtoG2. Peptide DD-018-WtoG2 does not co-elute with TNFα.

[0438] Example 38 Peptide TF-018-KtoR-WW and its oligomers were tested in TNFα / TNFR blocking assays and L-929 cell killing assays.

[0439] The activity of peptide TF-018-KtoR-WW (SEQ ID NO: 62) as a monomer, dimer, and trimer was determined in a TNFα / TNFR blocking assay (Example 3) and an 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

[0440] In addition, the activity of the peptide monomers and trimers was compared with an anti-TNFα antibody (R&D Systems (research grade)).

[0441] Figure 22A shows a comparison of the binding affinity of the monomeric, dimeric, and trimeric peptide TF-018-KtoR-WW in a TNFα / TNFR blocking assay. Multimerization of the peptides increased the IC 50 Figure 22B shows a comparison of the monomeric and trimeric peptides TF-018-KtoR-WW with an anti-TNFα antibody (R&D Systems (research grade)). The IC of the trimeric peptide TF-018-KtoR-WW 50 was better than that of anti-TNFα antibodies in the TNFα / TNFR blocking assay.

[0442] Figure 23A shows a comparison of the activity of the monomeric, dimeric, and trimeric peptide TF-018-KtoR-WW in an L-929 cell killing assay. 50 Figure 23B shows a comparison of the monomeric and trimeric peptides TF-018-KtoR-WW with an anti-TNFα antibody (R&D Systems (research grade)). The EC of the trimeric peptide TF-018-KtoR-WW was 50 The values ​​were better than those of the anti-TNFα antibody in this assay.

[0443] Example 39 Binding affinity determination of peptide TF-018-KtoR-WW monomer and trimer

[0444] The binding affinity of peptide TF-018-KtoR-WW (SEQ ID NO: 62) monomer and trimer was determined by surface plasmon resonance (SPR). Recombinant biotin-TNFα was immobilized on the SPR chip surface by interaction with a neutravidin-modified chip. Different concentrations of the monomer or trimer were then introduced across the surface, and relative binding parameters were determined. The peptide TF-018-KtoR-WW monomer had an affinity of approximately 50 nM, determined using monomer concentrations of 1200 nM, 625 nM, and 312.5 nM. The affinity of peptide TF-018-KtoR-WW C-trimer could not be determined in this assay (using concentrations of 3.75 nM and 1.88 nM) due to its strong interaction with immobilized TNFα.

[0445] Example 40 The peptide TF-18-KtoR-WW C-trimer is able to block the activity of membrane-bound TNFα

[0446] The peptide TF-18-KtoR-WW C-trimer was tested for its ability to specifically block the activity of membrane-bound TNFα using the L-929 cell killing assay. HEK293T cells express either wild-type (WT) or membrane-restricted (Δ1-12 aa) TNFα (see Ruuls et al., Immunity 15:533-543, 2001; Decoster et al., J. Biol. Chem. 270:18473-18478, 1995). The membrane-restricted form of TNFα lacks the substrate cleavage site (Δ1-12 aa) for TACE (TNFα-converting enzyme). To assess the blocking activity of TF-18-KtoR-WW C-trimer against membrane-associated TNFα, L-929 (TNFR-expressing) cells were overlaid against HEK293T cell lines (expressing either WT or membrane-restricted (Δ1-12 aa) TNFα) in the absence or presence of the trimer.

[0447] Referring to Figure 24, in the first (left-most) panel, addition of WT TNFα-expressing cells resulted in L-929 cell death. In the second panel (from the left), addition of membrane-restricted TNFα (Δ1-12aa TNFα)-expressing cells resulted in L-929 cell death. In the third panel (from the left), addition of WT TNFα-expressing cells and TF-18-KtoR-WW C-trimer rescued L-929 cell death, depending on the amount of TNFα-containing cells added. In the fourth panel (right-most panel), addition of TNFα (Δ1-12aa TNFα)-expressing cells and TF-18-KtoR-WW C-trimer rescued L-929 cell death.

[0448] In conclusion, TF-18-KtoR-WW C-trimer was able to rescue TNFα-mediated cell killing for both WT and membrane-restricted TNFα.

[0449] Example 41 Stability of the peptide TF-18-KtoR-WW C-trimer in simulated gastric and intestinal fluids

[0450] 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. The simulated gastric fluid (SGF - RICCA Chemical Company, Product No. 7108) contained 0.7% (v / v) hydrochloric acid and 0.2% (w / v) sodium chloride in 3.2 mg of freshly added purified pepsin (derived from porcine gastric mucosa, with an activity of 800-2500 units per mg of protein) per mL of solution. The pH of the simulated gastric fluid was 1-1.6. Simulated intestinal fluid (SIF-RICCA Chemical Company, product number 7109) contained 0.68% (w / v) potassium phosphate, 0.06% (w / v) sodium hydroxide, and 1% (w / v) pancreatin. The pH of this solution was 6.7–6.9.

[0451] 26 μM of the TF-18-KtoR-WW peptide C-trimer was incubated in SGF and SIF at 37°C for 24 hours, followed by HPLC. The amount of TF-18-KtoR-WW peptide C-trimer remaining after 24 hours was determined by comparing peak areas. The L-amino acid form of the TF-18-KtoR-WW peptide C-trimer was used as a control. The TF-18-KtoR-WW peptide C-trimer was stable under these conditions, with only minimal degradation evident after 24 hours, whereas the L-amino acid form of the TF-18-KtoR-WW peptide C-trimer was largely degraded within 8 hours or 1 hour in SGF and SIF, respectively.

[0452] Example 42 Whole blood cytokine release assay.

[0453] The effect of the peptide TF-18-KtoR-WW C-trimer on IL-8 release in stimulated human whole blood ex vivo was evaluated. Three stimuli were included in the study: recombinant human TNF (CellSystems #CS-C1140), LPS (Invivogen #REP-HEBS-10), and anti-CD3 and anti-CD28 antibodies (ThermoFisher #16-0037-81 and #16-0289-81).

[0454] Whole blood assays were 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 sample collection, French National Blood Service) was anticoagulated with heparin.

[0455] 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 blood stimulated with TNF, LPS, or anti-CD3 and anti-CD28 antibodies in the absence of compound. Stimulation of whole blood was performed in a U-bottom 96-well plate as follows. For stimulation with TNF, the peptide TF-18-KtoR-WW C-trimer and TNF (10 ng / mL) were preincubated at room temperature for 30 minutes before being added to the diluted blood, which was then further incubated at 37°C and 5% CO2 for 24 hours. For stimulation with LPS (0.1 EU / mL) or anti-CD3 and anti-CD28 antibodies (0.1 μg / mL anti-CD3 and 1 μg / mL anti-CD28), the peptide TF-18-KtoR-WW C-trimer was first mixed with the diluted blood, and then the stimuli were added according to the plate layout. Controls were prepared in a similar manner. Blood samples were incubated in RPMI containing stabilized L-glutamine at 37°C and 5% CO2. After 24 hours of incubation at 37°C and 5% CO2, supernatants were collected and stored at -20°C. The final reaction volume was 300 μL per well, and the final blood concentration was 50%. IL-8 released into the culture supernatant was measured using a specific ELISA kit (Human IL-8 ELISA Kit, R&D Systems, #DY208) according to the supplier's instructions.

[0456] After 24 hours of incubation, no signs of hemolysis were observed in the collected supernatants. In the absence of stimulation, IL-8 levels averaged 450–1070 pg / mL. Incubation of blood with 100 nM of the TF-18-KtoR-WW C-trimer peptide did not induce IL-8 production by itself. A decrease in spontaneous IL-8 release was observed in the presence of 100 nM of the TF-18-KtoR-WW C-trimer peptide. In the presence of the TF-18-KtoR-WW C-trimer peptide, a clear dose-dependent decrease in IL-8 released by stimulated blood was observed in three donors. The degree of IL-8 inhibition by the TF-18-KtoR-WW C-trimer peptide varied among the three stimuli. TNF-induced IL-8 was completely inhibited by 100 nM (and 10 nM for donor 2) of the peptide TF-18-KtoR-WW C-trimer. LPS-induced IL-8 was partially inhibited in blood from donors 1 and 3 but not in blood from donor 2. Anti-CD3 antibody and anti-CD28 antibody-induced IL-8 were also completely (donors 1 and 3) or partially (donor 2) inhibited in the presence of the peptide TF-18-KtoR-WW C-trimer. Referring to Figure 25, the average IL-8 inhibition of triplicate samples for each condition is shown.

[0457] Example 43 The peptide TF-18-KtoR-WW C-trimer promotes survival in vivo in a human TNFα mouse challenge model.

[0458] Healthy normal mice were cultured at time zero (T=0) and LD 80Mice were challenged with 100 mg of human TNFα (HuTNFα) and monitored for 24 hours. Thirty minutes before challenge, mice were pretreated with an anti-TNFα monoclonal antibody (research-grade adalimumab; R&D Systems) or the peptide TF-18-KtoR-WW C-trimer at a 1x or 5x huTNFα challenge dose. Mice were treated again with the peptide TF-18-KtoR-WW C-trimer at 0.5 and 1.5 hours. Referring to Figure 26, the anti-TNFα monoclonal antibody (anti-TNF) inhibits both mouse and human TNF-alpha, while the peptide TF-18-KtoR-WW C-trimer inhibits only human TNF-alpha.

[0459] Example 44 Single-dose subcutaneous administration of peptide TF-18-KtoR-WW C-trimer

[0460] Mice were dosed with peptide TF-18-KtoR-WW C-trimer diluted in PBS and administered as a single subcutaneous dose in two male and two female CD1 mice. Referring to Figures 27 and 28, the average amount of peptide TF-18-KtoR-WW C-trimer in plasma is shown for each group of four animals, two males and two females. Panels A and B show two different graphical representations of the data.

[0461] Example 45 Peptide TF-18-KtoR-WW C-trimer plasma levels after oral administration

[0462] The peptide TF-18-KtoR-WW C-trimer was diluted to 50 mg / kg in PBS and administered to two male and two female mice by single oral gavage before serum collection. See Table 1 below for the concentrations (ng / mL) of peptide TF-18-KtoR-WW C-trimer in the serum of all four animals at 6, 12, and 24 hours post-dosing. Following oral administration, the peptide TF-18-KtoR-WW C-trimer was detectable in the plasma of all four animals 24 hours post-dosing. [Table 1]

[0463] Example 46 Oral administration of peptide TF-18-KtoR-WW C-trimer

[0464] The peptide TF-18-KtoR-WW C-trimer was administered intravenously (5 mg / kg IV), subcutaneously (50 mg / kg SC), and orally (100 mg / kg PO) to C57Bl / 6 mice (3 animals per group). The vehicle was 91% sodium phosphate buffer (20 mM) and 9% sucrose. The peptide TF-18-KtoR-WW C-trimer concentration (nM) was measured in plasma or kidney (in the case of 50 mg / kg SC dosing) by mass spectrometry. Briefly, intestinal, liver, and kidney tissues were thoroughly flushed of luminal contents before homogenization and analysis. The peptide TF-18-KtoR-WW C-trimer was extracted from serum by precipitation of serum components using an organic solvent (acetonitrile crash), separated from remaining contaminating proteins by C18 reverse-phase HPLC at 40 °C, and the peptide was identified by ESI-MS. Peptide TF-18-KtoR-WW C-trimer levels were quantified by comparing the signal of unknown samples to that of a standard curve and quality control samples. All samples were spiked with a known amount of a "heavy" internal standard to control for variability in extraction, injection, and ionization.

[0465] Referring to Table 2 and Figure 29, peptide TF-18-KtoR-WW C-trimer was detected in plasma after administration of 5.0 mg / kg IV or 50 mg / kg SC. It was also detected in the kidney after 50 mg / kg SC dosing, with 10 percent of the initial exposure present in the kidney 24 hours later. The long terminal half-life of peptide TF-18-KtoR-WW C-trimer was 18 hours after intravenous administration.

[0466] Referring to Table 3 and Figure 30, oral (PO) dosing (100 mg / kg) resulted in low amounts of peptide TF-18-KtoR-WW C-trimer detectable in the liver and kidney (in two of three animals), indicating that some peptide TF-18-KtoR-WW C-trimer entered the systemic circulation. Plasma exposure was measurable in one animal per group. Peptide TF-18-KtoR-WW C-trimer was detectable in the liver and kidney, an indication of systemic distribution after oral delivery. In addition, high concentrations of peptide TF-18-KtoR-WW C-trimer were observed in small intestinal and large intestinal tissues. [Table 2] [Table 3]

[0467] Example 47 LPS challenge model

[0468] Tg1278 (mTNF KO / hTNF KI) mice are a transgenic strain with normally regulated and expressed human TNFα (hTNFα) in the absence of mouse TNFα. 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 serum was collected for analysis of IL-6 production. IL-6 was detected by ELISA. Referring to Figure 31, subcutaneous administration of the peptide TF-18-KtoR-WW C-trimer at 5, 15, and 50 mg / kg per mouse one hour before LPS treatment resulted in complete inhibition of TNF-mediated IL-6 production at all doses. The activity of peptide TF-18-KtoR-WW C-trimer was comparable to that of research-grade etanercept (labeled Enbrel in the figure) at a dose of 10 mg / kg per mouse administered 16 hours before LPS treatment. Referring to Figure 32, peptide TF-18-KtoR-WW C-trimer was able to inhibit the production of mouse KC (chemokine (C-X-C motif) ligand 1 (CXCL1)) in a similar manner.

[0469] Various publications, including patents, patent application publications, and scientific articles, are cited herein, the disclosures of which are incorporated by reference in their entireties for all purposes.

[0470] While illustrative embodiments have been shown and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. Table 4. Disclosed peptide sequences SEQ ID NO: 1 CX 2 -X 3 -X 4 -X 5 -X 6 -FFNX 10 -X 11 -C (where X 2 From X 6 , X 10each of which is a D-amino acid or a D-α-amino acid analog thereof; X 2 is the D-form of any of Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), Ile (I), Met (M), or Trp (W), or a D-α-amino acid analog thereof; X 3 is the D-form of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H) or Leu (L), or a D-α-amino acid analog thereof; X 4 is a polar amino acid, including the D-form of 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-α-amino acid analog thereof; X 5 is the D-form of Pro, Trp, His, Gln, Gly, Arg, Ala, Val, or Leu, or a D-α-amino acid thereof; X 6 is the D-form of Arg (R), His (H), Lys (K), Glu (E), Gln (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-α-amino acid analog thereof; X 10 is the D-form of Trp (W) or Tyr (Y), or a D-α-amino acid analog thereof; X 11 is the D-form of Trp (W), Tyr (Y), Gln (Q) or His (H), or a D-α-amino acid analog thereof; C represents the D-form of cysteine, F represents the D-form of phenylalanine, and N represents the D-form of asparagine or the D-α-amino acid analogs of C, F, or N. SEQ ID NO: 2 C * -X 2 -X 3 -X 4 -X 5 -X 6 -FNNX 10 -X11 -C * (SEQ ID NO: 2) (wherein X 1 From X 6 , X 10 and X 11 are each a D-amino acid or a Dα-amino acid analog thereof, and C, F, and N are the D-forms of cysteine, phenylalanine, and asparagine, or D-α-amino acid analogs thereof; X 2 is the D-form of Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), Ile (I), Met (M), or Trp (W), or a D-α-amino acid analog thereof; X 3 is selected from the D-form of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H) or Leu (L), or a D-α-amino acid analog thereof; X 4 is a polar amino acid selected from the D-form of 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-α-amino acid analog thereof; X 5 is the D-form of Pro (P), Trp (W), His (H), Gln (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), or a D-α-amino acid analog thereof; X 6 is the D-form of Arg (R), His (H), Lys (K), Glu (E), Gln (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-α-amino acid analog thereof; X 10 is the D-form of Trp (W) or Tyr (Y), or a D-α-amino acid analog thereof; X 11 is the D-form of Trp (W), Tyr (Y), Gln (Q) or His (H), or a D-α-amino acid analog thereof; *represents an optional intramolecular disulfide bond; C represents the D-form of cysteine ​​or its D-α-amino acid analog; F represents the D-form of phenylalanine or its D-α-amino acid analog; and N represents the D-form of asparagine or its D-α-amino acid analog). SEQ ID NO: 3 C * -X 2 -[W / F / Y]-X 4 -X 5 -X 6 -FNN-[W / Y]-WC * (SEQ ID NO: 3) (X 2 is the D-form of Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), Ile (I), Met (M), or Trp (W), or a D-α-amino acid analog thereof; X 4 is a polar amino acid selected from the D-form of 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-α-amino acid analog thereof; X 5 is the D-form of Pro (P), Trp (W), His (H), Gln (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), or a D-α-amino acid analog thereof; X 6 is the D-form of Arg (R), His (H), Lys (K), Glu (E), Gln (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-α-amino acid analog thereof; *represents an optional intramolecular disulfide bond; C represents the D-form of cysteine ​​or its D-α-amino acid analog; F represents the D-form of phenylalanine or its D-α-amino acid analog; W represents the D-form of tryptophan or its D-α-amino acid analog; F represents the D-form of phenylalanine or its D-α-amino acid analog; Y represents the D-form of tyrosine or its D-α-amino acid analog; and N represents the D-form of asparagine or its D-α-amino acid analog). [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] SEQ ID NO: 111 CX 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10-X 11 -C (where X 2 From X 11 each of which is a D-amino acid; 2 is the D-form of any of the common L-amino acids other than 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), Gln (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G); X 5 is the D-form of any of the common L-amino acids other than cysteine; X 6 is the D-form of any of the common L-amino acids other than cysteine; X 7 is the D form of Phe(F); X 8 is the D-type of Asn(N), and X 9 is the D-type of Asn(N), and X 10 is the D-form of Trp (W) or Tyr (Y); X 11 is the D-form of Trp (W), Gln (Q), Tyr, or His (H); C represents the D-form of 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 * (where X 2 From X 11 each of which is a D-amino acid; 2 is the D-form of any of the common L-amino acids other than cysteine; X 3 are the D forms of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H), and Leu (L); X 4are 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 is the D-form of any of the common L-amino acids other than cysteine; X 6 is the D-form of any of the common L-amino acids other than cysteine; X 7 is the D form of Phe(F); X 8 is the D-type of Asn(N), and X 9 is the D-type of Asn(N), and X 10 is the D-form of Trp (W) and Tyr (Y); X 11 is the D-form of Trp (W), Tyr (Y), Gln (Q) or His (H); C represents the D-form of cysteine; * represents an intramolecular bond, if necessary) SEQ ID NO: 113 C * -X 2 -[W / F / Y]-[Polarity]-X 5 -X 6 -FNN-[W / Y]-WC * (SEQ ID NO: 3) (wherein X 2 is the D form of any of the standard L-amino acids other than Cys, and X 5 is the D-form of any of the standard L-amino acids other than cysteine, and X 6 represents the D-amino acid, which is the D-form of any of the standard L-amino acids other than cysteine, and polarity includes one of R, K, H, E, D, Q, N, T, S, P, A, or G; * indicates an intramolecular disulfide bond where appropriate) SEQ ID NO: 114 X * TFQPRFNNWWC * (where, * represents an optional intramolecular disulfide bond, and X is D-penicillamine. SEQ ID NO: 115 C * TFQPRFNNWX * (where, *represents an optional intramolecular disulfide bond, and X is D-penicillamine. The embodiments of the invention in which an exclusive property right or privilege is claimed are defined as follows:

Claims

1. 1. A D-peptide or salt thereof, said peptide comprising a core TNFα binding domain of D-amino acids and having 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), and X １ From X ６ , X １０ and X １１ each of which is a D-amino acid or a D-α-amino acid analog thereof; a.X ２ is the D-form of Thr (T), Val (V), His (H), Leu (L), Gln (Q), Ala (A), Ile (I), Met (M), or Trp (W), or a D-α-amino acid analog thereof; b. X ３ are the D-forms of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H) and Leu (L), or D-α-amino acid analogs thereof; c. X ４ are polar amino acids, including the D-forms of 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; d. X ５ is the D-form of Pro (P), Trp (W), His (H), Gln (Q), Arg (R), Ala (A), Val (V), Leu (L), or Gly (G), or a D-α-amino acid analog thereof; e. X ６ is the D-form of Arg (R), His (H), Lys (K), Glu (E), Gln (Q), Val (V), Leu (L), Ser (S), or Ala (A), or a D-α-amino acid analog thereof; f.X １０ is the D-form of Trp (W) or Tyr (Y), or a D-α-amino acid analog thereof; g. X １１ is the D-form of Trp (W), Tyr (Y), Gln (Q), or His (H), or a D-α-amino acid analog thereof; k. A D-peptide or a salt thereof, wherein C represents the D-form of cysteine ​​or a D-α-amino acid analog thereof; N represents the D-form of asparagine or a D-α-amino acid analog thereof; and F represents the D-form of phenylalanine or a D-α-amino acid analog thereof.

2. The core TNFα binding domain has the following amino acid sequence: C-X ２ -[W / F / Y]-X ４ -X ５ -X ６ 2. The D-peptide of claim 1, having the structure -FNN-[W / Y]-WC (SEQ ID NO: 3), or a salt thereof.

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

4. a.X ２ is the D-form of Thr, Val, His, Leu, Gln, Ala, lie, Met, or Trp, or a D-α-amino acid analog thereof; b. X ２ is the D-form of Thr, Val, His, Leu, or Gln, or a D-α-amino acid analog thereof; c. X ２ is the D-form of Thr, Val, His, or Leu, or a D-α-amino acid analog thereof; d. X ２ is the D-form of Thr, Val, or His, or a D-α-amino acid analog thereof; e. X ２ is the D-form of Thr or Val, or a D-α-amino acid analog thereof; f.X ２ is the D-form of Thr, or a D-α-amino acid analog thereof; or g. X ２ The D-peptide or salt thereof according to any one of claims 1 to 3, wherein is the D-form of Val or a D-α-amino acid analog thereof.

5. a.X ３ is the D-form of Trp, Phe, Tyr, or Ser, or a D-α-amino acid analog thereof; b. X ３ is the D-form of Trp, Phe, or Tyr, or a D-α-amino acid analog thereof; c. X ３ is the D-form of Trp or Phe, or a D-α-amino acid analog thereof; d. X ３ is the D-form of Trp or a D-α-amino acid analog thereof; or e. X ３ The D-peptide or salt thereof according to any one of claims 1 or 3 to 4, wherein is the D-form of Phe or a D-α-amino acid analog thereof.

6. a.X ４ is the D-form of Arg, His, Gln, Asn, Lys, Thr, or Ser, or a D-α-amino acid analog thereof; b. X ４ is the D-form of Arg, His, Gln, or Asn, or a D-α-amino acid analog thereof; c. X ４ is the D-form of Arg, His, or Gln, or a D-α-amino acid analog thereof; d. X ４ is the D-form of Arg, Gln, or Asn, or a D-α-amino acid analog thereof; or e. X ４ The D-peptide or salt thereof according to any one of claims 1 to 5, wherein is the D-form of Gln or a D-α-amino acid analog thereof.

7. a.X ５ is the D-form of Pro, Trp, His, Gln, Gly, Arg, Ala, Val, or Leu, or a D-α-amino acid analog thereof; b. X ５ is the D-form of Pro, Trp, His, Gln, Gly, Arg, or Val, or a D-α-amino acid analog thereof; c. X ５ is the D-form of Pro, Trp, or His, or a D-α-amino acid analog thereof; d. X ５ is the D-form of Pro or Trp, or a D-α-amino acid analog thereof; or e. X ５ The D-peptide or salt thereof according to any one of claims 1 to 6, wherein is the D-form of Pro or a D-α-amino acid analog thereof.

8. a.X ６ is the D-form of Arg, His, Lys, Glu, Gln, Val, Leu, Ser, or Ala, or a D-α-amino acid analog thereof; b. X ６ is the D-form of Arg, His, Lys, Glu, Gln, Val, or Leu, or a D-α-amino acid analog thereof; c. X ６ is the D-form of Arg, His, Lys, Glu, or Gln, or a D-α-amino acid analog thereof; d. X ６ is the D-form of Arg, His, Lys, or Glu, or a D-α-amino acid analog thereof; e. X ６ is the D-form of Arg, Lys, or His, or a D-α-amino acid analog thereof; f.X ６ is the D-form of Arg, or a D-α-amino acid analog thereof; g. X ６ is the D-form of Lys, or a D-α-amino acid analog thereof; or h.X ６ The D-peptide or salt thereof according to any one of claims 1 to 7, wherein is the D-form of His or a D-α-amino acid analog thereof.

9. a.X １０ is the D-form of Trp, or a D-α-amino acid analog thereof; or b. X １０ 9. The D-peptide or salt thereof of any one of claims 1 and 3 to 8, wherein is the D-form of Tyr or a D-α-amino acid analog thereof.

10. a.X １１ is the D-form of His (H), Trp (W), Tyr (Y), or Gln (Q), or a D-α-amino acid analog thereof; b. X １１ is the D-form of Tyr (Y), or a D-α-amino acid analog thereof; or c. X １１ 10. The D-peptide or salt thereof of any one of claims 1 and 3 to 9, wherein is the D-form of Trp(W) or a D-α-amino acid analog thereof.

11. X １ From X ６ , X １０ , and X １１ The D-peptide or salt thereof according to any one of claims 1 to 10, wherein each of

12. The core TNFα binding domain has the amino acid sequence: 【Chemistry 49】 [Transformation 50] The D-peptide or salt thereof according to any one of claims 1 to 11, having the formula:

13. 10. The D-peptide or salt thereof of any of the preceding claims, wherein the core TNFα binding domain has the amino acid sequence set forth in SEQ ID NOs: 77-110.

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

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

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

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

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

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

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

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

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

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

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

25. 25. The D-peptide or salt thereof of claim 22 or 24, wherein the PEG group is attached to the N-terminus of the D-peptide.

26. 25. The D-peptide or salt thereof of claim 22 or 24, wherein the PEG group is attached to the C-terminus of the D-peptide.

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

29. A multimer of a D-peptide according to any one of the preceding claims, or a salt thereof.

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

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

32. 32. The multimer or salt thereof of any one of claims 29 to 31, further comprising a multimeric scaffold attached to said D-peptide, optionally via a linker.

33. 33. The multimer or salt thereof of claim 32, wherein the multimeric scaffold is trimeric.

34. 33. The multimer or salt thereof of claim 32, wherein the multimeric scaffold is a tetramer.

35. Trifunctional crosslinkers include tris(succinimidyl)aminotriacetate (TSAT), tris-succinimidyl(6-aminocaproyl)aminotriacetate (LC-TSAT), and the 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, Fmoc scaffold with PEG27 chain, cyclohexa scaffold (tris(2,5-dioxopyrrolidin-1-yl)cyclohexane-1,3,5-tricarboxylate), nitro scaffold (bis(2,5-dioxopyrrolidin-1-yl)4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl) 35. The multimer or salt thereof of claim 33 or 34, wherein the multimer is selected from the group consisting of: an amine scaffold (bis(2,5-dioxopyrrolidin-1-yl)4-amino-4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)heptanedioate), an amine-PEG27 scaffold, a cholesterol scaffold, a heterotetrameric PEG scaffold based on a 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propionic acid scaffold, a multimeric scaffold based on 4-amino-4-(2-carboxyethyl)heptanedioic acid, or 3-{2-amino-3-(2-carboxyethoxy)-2-[(2-carboxyethoxy)methyl]propoxy}propionic acid.

36. The multimeric peptide construct comprises: 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 -cyclohexa; i. [Peptide-PEG12-K-amide] ３ - nitro; j. [Peptide-PEG12-K-amide] ３ -PEG27-amine; k. [Peptide-PEG12-K-amide] ３ -amines; l. [Peptide-PEG12-K-amide] ３ -PEG27-biotin; or m. [Peptide-PEG12-K-amide] ３ -PEG27-cholesterol; The multimer or salt thereof according to claim 35, wherein the peptide is a D-peptide.

37. below: 【Chemistry 51】 29. The D-peptide or salt thereof according to any one of claims 1 to 28, having at least one amino acid sequence selected from the group consisting of:

38. below: 【Chemistry 52】 【Chemistry 53】 The multimer or salt thereof according to any one of claims 29 to 36, comprising a D-peptide having at least one amino acid sequence selected from the group consisting of:

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

40. 40. The pharmaceutical composition of claim 39, formulated for parenteral administration.

41. 41. The pharmaceutical composition of claim 40, formulated for intravenous, intramuscular, or subcutaneous administration.

42. 40. The pharmaceutical composition of claim 39, formulated for oral administration.

43. 40. The pharmaceutical composition of claim 39, formulated for topical administration.

44. 44. The pharmaceutical composition of claim 43, formulated for topical administration to the skin (transdermally) or eye.

45. 40. The pharmaceutical composition of claim 39, formulated for rectal administration.

46. A lyophilized composition comprising at least one D-peptide or multimeric peptide construct according to any one of claims 1 to 38, or a pharmaceutically acceptable salt thereof, and a stabilizing agent.

47. A freeze-dried composition of the pharmaceutical composition according to any one of claims 39 to 45 and a stabilizer.

48. 48. A rewetting solution for a freeze-dried composition according to any one of claims 46 or 47.

49. 46. ​​A method of treating a TNFα mediated disease, comprising administering an effective amount of a D-peptide according to any one of claims 1 to 28 or 37, a multimeric peptide construct according to any one of claims 29 to 36 and 38, or a pharmaceutical composition according to any one of claims 39 to 45, or a pharmaceutically acceptable salt thereof.

50. 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 (intermediate uveitis, posterior uveitis, or panuveitis), or spondyloarthritis not meeting radiographic criteria.

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

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

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

54. 53. The method of claim 51 or 52, wherein the administration is rectal.

55. 53. The method of claim 51 or 52, wherein the administration is parenteral.

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

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

58. 58. The method of claim 56 or 57, wherein the administration is topical.

59. 58. The method of claim 56 or 57, wherein the administration is parenteral.

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

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

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

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

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

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

66. 66. The method of any one of claims 63 to 65, wherein said administration is by oral administration, parenteral administration, topical (transdermal) administration, or rectal administration.

67. 66. The method of any one of claims 63 to 65, wherein the D-peptide or multimeric peptide construct thereof, or a pharmaceutically acceptable salt thereof, is administered locally to reduce TNFα activity or inflammation or an inflammatory response.

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

69. 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 (intermediate uveitis, posterior uveitis, or panuveitis), or spondyloarthritis not meeting radiographic criteria.

70. 39. A D-peptide or multimeric peptide construct thereof according to any one of claims 1 to 38, or a pharmaceutically acceptable salt thereof, for use as a medicine.

71. 49. A D-peptide or multimeric peptide construct thereof according to any one of claims 1 to 38, or a pharmaceutically acceptable salt thereof, for use in a method of therapeutically treating a subject.

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

73. 73. The D-peptide of 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 (intermediate, posterior, or panuveitis), or spondyloarthritis not meeting radiographic criteria.

74. 1. A D-peptide or salt thereof, said peptide comprising a core TNFα binding domain of D-amino acids and having 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: 111), and X ２ From X １１ each of X is a D-amino acid; ２ is the D form of any of the common L-amino acids 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), Gln (Q), Asn (N), Thr (T), Ser (S), Pro (P), Ala (A), or Gly (G); X ５ is the D form of any of the common L-amino acids except cysteine; X ６ is the D form of any of the common L-amino acids except cysteine; X ７ is the D-form of Phe(F); X ８ is the D-form of Asn(N), and X ９ is the D-form of Asn(N), and X １０ is the D form of Trp (W) or Tyr (Y); X １１ is the D-form of Trp (W), Gln (Q), Tyr, or His (H); C represents the D-form of cysteine, or a salt thereof.

75. 1. A D-peptide or salt thereof, wherein the peptide comprises a core TNFα binding domain of D-amino acids and has the following amino acid sequence: ＊ -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), and X ２ From X １１ each of X is a D-amino acid; ２ is the D form of any of the common L-amino acids except cysteine; X ３ are the D forms of Trp (W), Phe (F), Tyr (Y), Ser (S), His (H) and Leu (L); X ４ 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 is the D form of any of the common L-amino acids except cysteine; X ６ is the D form of any of the common L-amino acids except cysteine; X ７ is the D-form of Phe(F); X ８ is the D-form of Asn(N), and X ９ is the D-form of Asn(N), and X １０ is the D form of Trp (W) and Tyr (Y); X １１ is the D-form of Trp (W), Tyr (Y), Gln (Q) or His (H); C represents the D-form of cysteine; ＊ represents an optional intramolecular bond, a D-peptide or a salt thereof.

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

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