阅读说明：本技术 调节γC-细胞因子信号传导对于治疗脱发和脱发相关病症的影响 (Effect of modulation of gamma C-cytokine signaling on the treatment of alopecia and alopecia-related disorders ) 是由 田谷丰 纳兹利·阿兹米 于 2020-04-30 设计创作，主要内容包括：γc-家族细胞因子,即白介素-2(IL-2)、白介素-4(IL-4)、白介素-7(IL-7)、白介素-9(IL-9)、白介素-15(IL-15)和白介素-21(IL-21)与重要的人类疾病(例如脱发和脱发相关病症)相关。描述了调节至少一个γc-细胞因子家族成员的信号传导以抑制、改善、降低至少一种脱发相关病症的严重程度,治疗至少一种脱发相关病症,延迟至少一种脱发相关病症的发作,或者预防至少一种脱发相关病症的组合物、方法和试剂盒。(The yc-family cytokines, namely interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-15 (IL-15), and interleukin-21 (IL-21), are associated with important human diseases, such as alopecia and alopecia-related disorders. Compositions, methods, and kits are described that modulate signaling by at least one member of the yc-cytokine family to inhibit, ameliorate, reduce the severity of, treat, delay the onset of, or prevent at least one alopecia-related disorder.)

1. A composition comprising:

a therapeutic compound in an amount sufficient to modulate signaling by at least one member of the yc-cytokine family, thereby inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing at least one alopecia-related disorder; and

a pharmaceutically acceptable carrier.

2. The composition according to claim 1, wherein the at least one alopecia-related disorder is selected from alopecia, pemphigus, pemphigoid, psoriasis, vitiligo, graft versus host disease, and immune-mediated alopecia.

3. The composition of claim 1, wherein the at least one yc-cytokine family member is selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

4. The composition of claim 1, wherein the therapeutic compound is at least one of a yc-cytokine antagonist peptide, a yc-cytokine antagonist peptide derivative, an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, or a combination thereof.

5. The composition of claim 4, wherein the yc-cytokine antagonist peptide comprises a partial sequence of the yc-box D-helical region of each of at least two yc-cytokine family members.

6. The composition of claim 5, wherein the partial sequence comprises a contiguous block of at least 5 amino acids of the yc-box D-helical region of each of the at least two yc-cytokine family members.

7. The composition of claim 5, wherein the partial sequence comprises a contiguous block of 1-10 amino acids of the yc-box D-helical region of each of the at least two yc-cytokine family members.

8. The composition of any one of claims 5-7, wherein the yc-box D-helical region of each of the at least two yc-cytokine family members is selected from the group consisting of IL-15, IL-2, IL-21, IL-4, IL-9, and IL-7.

9. The composition of any one of claims 4-8, wherein the yc-cytokine antagonist peptide comprises 11 to 50 amino acids.

10. The composition of any one of claims 4-9, wherein the yc-cytokine antagonist peptide further comprises a conjugate at the N-terminus, C-terminus, side chain residues, or a combination thereof.

11. The composition of claim 10, wherein the conjugate comprises one or more additional moieties selected from the group consisting of: bovine Serum Albumin (BSA), albumin, Keyhole Limpet Hemocyanin (KLH), Fc region of IgG, biological protein functioning as a scaffold, antibody against cell-specific antigen, receptor, ligand, metal ion, and polyethylene glycol (PEG).

12. The composition of any one of claims 4-11, wherein the yc-cytokine antagonist peptide further comprises a signal peptide.

13. The composition of any one of claims 4-12, wherein said yc-cytokine antagonist peptide comprises the amino acid sequence D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO:2), wherein X represents any amino acid.

14. The composition of claim 4, wherein the yc-cytokine antagonist peptide derivative shares at least about 50% identity with the peptide of SEQ ID NO. 2.

15. The composition of claim 4, wherein the yc-cytokine antagonist peptide derivative shares at least about 90% identity with the peptide of SEQ ID NO. 2.

16. The composition of claim 4, wherein the yc-cytokine antagonist peptide derivative shares at least about 95% identity with the peptide of SEQ ID NO 2.

17. The composition of claim 4, wherein the yc-cytokine antagonist peptide comprises the sequence of SEQ ID NO 1 (BNZ- γ).

18. The composition of claim 4, wherein said yc-cytokine antagonist peptide consists of the sequence of SEQ ID NO 1.

19. The composition of claim 4, wherein said yc-cytokine antagonist peptide and said yc-antagonist peptide derivative have similar physicochemical properties but different biological activities.

20. The composition of claim 4, wherein the yc-cytokine antagonist peptide derivative shares at least about 50% identity with the peptide of SEQ ID NO 1.

21. The composition of claim 4, wherein the yc-cytokine antagonist peptide derivative shares at least about 90% identity with the peptide of SEQ ID NO. 1.

22. The composition of claim 4, wherein the yc-cytokine antagonist peptide derivative shares at least about 95% identity with the peptide of SEQ ID NO 1.

23. The composition of any one of claims 1-22, wherein the pharmaceutically acceptable carrier is formulated for topical, oral, and/or parenteral delivery.

24. The composition of any one of claims 1-22, wherein the pharmaceutically acceptable carrier is formulated for topical delivery.

25. The composition of any one of claims 1-22, wherein the pharmaceutically acceptable carrier is formulated for oral delivery.

26. The composition of any one of claims 1-22, wherein the pharmaceutically acceptable carrier is formulated for parenteral delivery.

27. A method of inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing at least one hair loss-related disorder, comprising:

administering to a subject in need thereof the composition of any one of claims 1-26,

thereby inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing at least one hair loss-related disorder.

28. The method according to claim 27, wherein the at least one alopecia-related disorder is selected from alopecia, pemphigus, pemphigoid, psoriasis, vitiligo, graft versus host disease, and immune-mediated alopecia.

29. A method of designing a yc-cytokine antagonist peptide and/or a derivative thereof configured to modulate and/or block signaling by at least one yc-cytokine family member that inhibits, ameliorates, reduces the severity of, treats, delays the onset of, or prevents at least one alopecia-associated disorder, the method comprising the steps of:

obtaining the amino acid sequence of at least one yc-cytokine family member from an amino acid sequence database using a computer,

assembling a yc-cytokine antagonist peptide and/or a derivative thereof based on the sequence of the at least one yc-cytokine family member,

wherein the yc-cytokine antagonist peptide and/or derivative thereof modulates and/or blocks signaling of the at least one yc-cytokine family member.

30. The method of claim 29, wherein the at least one yc-cytokine family member is selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

31. The method of claim 29, wherein the yc-cytokine antagonist peptide comprises a partial sequence of the yc-box D-helical region of each of at least two yc-cytokine family members.

32. The method of claim 31, wherein the sequence comprises a contiguous block of at least 5 amino acids of the yc-box D-helical region of each of the at least two yc-cytokine family members.

33. The method of claim 31, wherein the sequence comprises a contiguous block of 1-10 amino acids of the yc-box D-helical region of each of the at least two yc-cytokine family members.

34. The method according to any one of claims 31-33, wherein the yc-box D-helical region of each of the at least two yc-cytokine family members is selected from the group consisting of IL-15, IL-2, IL-21, IL-4, IL-9 and IL-7.

35. The method of any one of claims 29-34, wherein the yc-cytokine antagonist peptide comprises 11 to 50 amino acids.

36. The method of any one of claims 29-35, wherein the yc-cytokine antagonist peptide further comprises a conjugate at the N-terminus, C-terminus, side chain residues, or a combination thereof.

37. The method of any one of claims 29-36, wherein the yc-cytokine antagonist peptide further comprises a signal peptide.

38. The method of any one of claims 29-37, wherein said yc-cytokine antagonist peptide comprises the amino acid sequence D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO:2), wherein X represents any amino acid.

39. The method of any one of claims 29-38, wherein the yc-cytokine antagonist peptide derivative shares at least about 50% identity with the peptide of SEQ ID No. 2.

40. The method of any one of claims 29-38, wherein the yc-cytokine antagonist peptide derivative shares at least about 90% identity with the peptide of SEQ ID No. 2.

41. The method of any one of claims 29-38, wherein the yc-cytokine antagonist peptide derivative shares at least about 95% identity with the peptide of SEQ ID No. 2.

42. The method of any one of claims 29-38, wherein the yc-cytokine antagonist peptide comprises the sequence of SEQ ID NO:1 (BNZ- γ).

43. The method of any one of claims 29-38, wherein the yc-cytokine antagonist peptide consists of the sequence of SEQ ID No. 1.

44. The method of any one of claims 29-38, wherein the yc-cytokine antagonist peptide derivative shares at least about 50% identity with the peptide of SEQ ID No. 1.

45. The method of any one of claims 29-38, wherein the yc-cytokine antagonist peptide derivative shares at least about 90% identity with the peptide of SEQ ID No. 1.

46. The method of any one of claims 29-38, wherein the yc-cytokine antagonist peptide derivative shares at least about 95% identity with the peptide of SEQ ID No. 1.

47. The method of any one of claims 29-46, wherein the yc-cytokine antagonist peptide and derivatives thereof have similar physicochemical properties but different biological activities.

48. A kit for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing at least one alopecia-related disorder, comprising:

the composition of any one of claims 1-26.

49. The kit according to claim 48, wherein said at least one alopecia-related disorder is selected from the group consisting of alopecia, pemphigus, pemphigoid, psoriasis, vitiligo, graft versus host disease, and immune-mediated alopecia.

FIELD

The present embodiments relate to inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing autoimmune diseases (e.g., hair loss and hair loss related disorders) by modulating signaling of at least one c-cytokine family member using one or more therapeutic compounds.

Description of the related Art

Cytokines are a diverse group of soluble factors that mediate a variety of cellular functions, such as growth, functional differentiation, and promotion or prevention of programmed cell death (apoptotic cell death). Unlike hormones, cytokines are not produced by specialized glandular tissues, but can be produced by a variety of cell types such as epithelial cells, stromal cells, or immune cells.

The yc-family of cytokines is a group of mammalian cytokines that are produced primarily by epithelial cells, stromal cells, and immune cells, and control the normal and pathological activation of diverse lymphocyte sets. These cytokines are strictly necessary for the early development of T cells in the thymus and their homeostasis in the peripheral system.

Background

SUMMARY

In some embodiments, the composition comprises a therapeutic compound in an amount sufficient to modulate signaling by at least one member of the yc-cytokine family, thereby inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing at least one alopecia-associated disorder; and a pharmaceutically acceptable carrier.

In some embodiments of the composition, the at least one alopecia-related disorder is selected from the group consisting of alopecia, pemphigus, pemphigoid, psoriasis, vitiligo, graft versus host disease, and immune-mediated alopecia.

In some embodiments of the composition, the at least one yc-cytokine family member is selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

In some embodiments of the composition, the therapeutic compound is at least one of a yc cytokine antagonist peptide, a yc cytokine antagonist peptide derivative, an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, or a combination thereof.

In some embodiments of the composition, the yc cytokine antagonist peptide comprises a partial sequence of the yc-box D-helical region of each of at least two yc-cytokine family members.

In some embodiments of the composition, the partial sequence comprises a contiguous block of at least 5 amino acids of the yc-box D-helical region of each of the at least two yc-cytokine family members.

In some embodiments of the composition, the partial sequence comprises a contiguous block of 1-10 amino acids of the yc-box D-helical region of each of the at least two yc-cytokine family members.

In some embodiments of the composition, the yc-box D-helical region of each of the at least two yc-cytokine family members is selected from the group consisting of IL-15, IL-2, IL-21, IL-4, IL-9, and IL-7.

In some embodiments of the composition, the yc cytokine antagonist peptide comprises 11 to 50 amino acids.

In some embodiments of the composition, the yc cytokine antagonist peptide further comprises a conjugate at the N-terminus, C-terminus, side chain residues, or a combination thereof.

In some embodiments of the composition, the conjugate comprises one or more additional moieties selected from the group consisting of: bovine Serum Albumin (BSA), albumin, Keyhole Limpet Hemocyanin (KLH), Fc region of IgG, biological protein functioning as a scaffold, antibody against cell-specific antigen, receptor, ligand, metal ion, and polyethylene glycol (PEG).

In some embodiments of the composition, the yc cytokine antagonist peptide further comprises a signal peptide.

In some embodiments of the compositions, the yc cytokine antagonist peptide comprises the amino acid sequence D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO:2), wherein X represents any amino acid.

In some embodiments of the compositions, the yc cytokine antagonist peptide derivative shares at least about 50% identity with the peptide of SEQ ID No. 2.

In some embodiments of the compositions, the yc cytokine antagonist peptide derivative shares at least about 90% identity with the peptide of SEQ ID No. 2.

In some embodiments of the compositions, the yc cytokine antagonist peptide derivative shares at least about 95% identity with the peptide of SEQ ID No. 2.

In some embodiments of the composition, the yc cytokine antagonist peptide comprises the sequence of SEQ ID NO:1 (BNZ- γ).

In some embodiments of the composition, the yc cytokine antagonist peptide and the yc antagonist peptide derivative have similar physicochemical properties, but have different biological activities.

In some embodiments of the compositions, the yc cytokine antagonist peptide derivative shares at least about 50% identity with the peptide of SEQ ID No. 1.

In some embodiments of the compositions, the yc cytokine antagonist peptide derivative shares at least about 90% identity with the peptide of SEQ ID No. 1.

In some embodiments of the compositions, the yc cytokine antagonist peptide derivative shares at least about 95% identity with the peptide of SEQ ID No. 1.

In some embodiments of the composition, the pharmaceutically acceptable carrier is formulated for topical, oral, and/or parenteral delivery.

In some embodiments of the composition, the pharmaceutically acceptable carrier is formulated for topical delivery.

In some embodiments of the composition, the pharmaceutically acceptable carrier is formulated for oral delivery.

In some embodiments of the composition, the pharmaceutically acceptable carrier is formulated for parenteral delivery.

In some embodiments, a method of inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing at least one hair loss-related disorder comprises administering one or more compositions provided herein to a subject in need thereof, thereby inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing at least one hair loss-related disorder.

In some embodiments of the methods of inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing at least one hair loss-related disorder, the at least one hair loss-related disorder is selected from the group consisting of hair loss, pemphigus, pemphigoid, psoriasis, vitiligo, graft versus host disease, and immune-mediated hair loss.

In some embodiments, the method of designing a yc-cytokine antagonist peptide and/or a derivative thereof comprises the steps of: obtaining an amino acid sequence of at least one yc-cytokine family member from an amino acid sequence database using a computer, assembling a yc-cytokine antagonist peptide and/or a derivative thereof based on the sequence of the at least one yc-cytokine family member, wherein the yc-cytokine antagonist peptide and/or derivative thereof modulates and/or blocks signaling of the at least one yc-cytokine family member, the yc-cytokine antagonist peptide and/or derivative thereof is configured to modulate and/or block signaling by at least one yc-cytokine family member, inhibit, ameliorate, reduce the severity of, treat, delay the onset of, or prevent at least one alopecia-associated disorder.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the at least one yc-cytokine family member is selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide comprises a partial sequence of the yc-box D-helical region of each of at least two yc-cytokine family members.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the sequence comprises a contiguous block of at least 5 amino acids of the yc-box D-helical region of each of the at least two yc-cytokine family members.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or derivative thereof, the sequence comprises a contiguous block of 1-10 amino acids of the yc-box D-helical region of each of at least two yc-cytokine family members.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or derivative thereof, the yc-cassette D-helix region of each of the at least two yc-cytokine family members is selected from the group consisting of IL-15, IL-2, IL-21, IL-4, IL-9, and IL-7.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide comprises 11 to 50 amino acids.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide further comprises a conjugate at the N-terminus, the C-terminus, a side chain residue, or a combination thereof.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide further comprises a signal peptide.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide comprises the amino acid sequence D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO:2), wherein X represents any amino acid.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide derivative shares at least about 50% identity with the peptide of SEQ ID No. 2.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide derivative shares at least about 90% identity with the peptide of SEQ ID No. 2.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide derivative shares at least about 95% identity with the peptide of SEQ ID No. 2.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide comprises the sequence of SEQ ID NO:1 (BNZ- γ).

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide derivative shares at least about 50% identity with the peptide of SEQ ID No. 1.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide derivative shares at least about 90% identity with the peptide of SEQ ID No. 1.

In some embodiments of the methods of designing a yc-cytokine antagonist peptide and/or a derivative thereof, the yc-cytokine antagonist peptide derivative shares at least about 95% identity with the peptide of SEQ ID No. 1.

In some embodiments of the methods of designing yc-cytokine antagonist peptides and/or derivatives thereof, the yc-cytokine antagonist peptides and derivatives thereof have similar physicochemical properties, but have different biological activities.

In some embodiments, a kit for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing at least one hair loss-related disorder comprises one or more compositions provided herein.

In some embodiments of the kit, the at least one alopecia-related disorder is selected from the group consisting of alopecia, pemphigus, pemphigoid, psoriasis, vitiligo, graft versus host disease, and immune-mediated alopecia.

Brief Description of Drawings

FIG. 1A shows an alignment of the D-helical regions of human yc-cytokine family members.

FIG. 1B depicts the yc-box (SEQ ID NO:9) and IL-2/IL-15 box (SEQ ID NO:10) motifs that produce a consensus sequence around the D-helix region of the yc-cytokine.

FIG. 2 depicts a graphical representation of the biochemical properties of amino acids.

FIG. 3A shows the inhibition of IL-15 and IL-9 activity by BNZ-gamma in a PT-18 proliferation assay.

FIG. 3B shows a proliferation assay of CTLL-2 cells grown in the presence of IL-2 or IL-15 and 0, 0.1, 1 or 10 μ M BNZ- γ.

Figure 4 shows the inhibition of IL-15 mediated tyrosine-phosphorylation of STAT5 by BNZ- γ.

FIG. 5 shows circulating levels of human cytokines IL-2, IL-15 and IFN γ following transplantation of huPBMC to NSG mice.

Figure 6A shows that human CD8+ T cells from representative NSG mice fully expressed NKG2D (CD314) 4 weeks after huPBMC transplantation.

Figure 6B shows the expansion of NKG2A + human CD8+ T cells (boxes) in representative NSG mice from 1 to 4 weeks after transplantation of hupmc.

Figure 7A shows specific depletion of human CD8+ T cells after injection of anti-CD 8 antibody in representative NSG mice 4 weeks after huPBMC transplantation. The plot after injection of anti-CD 8AB is day 8 after antibody injection.

Figure 7B shows the average recovery of body weight (in grams of days) following human CD8+ T cell depletion mediated by anti-CD 8 antibody in three NSG mice treated with antibody 4 weeks after transplantation of hupmc.

Figure 7C shows anti-CD 8 antibody-mediated regrowth of body hair after human CD8+ T cell depletion in representative NSG mice 14 days post antibody injection and 42 days post huPBMC transplantation.

Figure 8 shows the positive phosphorylation of Jak3 and STAT 5in NKG2A + (+) but not NKG2A- (-) CD8+ T cells isolated from representative NSG mice 4 weeks after huPBMC transplantation, indicating constitutive activation of γ C-cytokine signaling.

Figure 9A shows a positive correlation between the expansion of NKG2A + human CD8+ T cells from three representative humanized NSG mice and the levels of inflammatory cytokines IFN γ and yc-cytokines IL-2 and IL-15 over the course of 1 to 6 weeks post huPBMC transplantation.

Figure 9B shows that effective depletion of human NKG2A + CD8+ T cells resulted in improvement of GvHD symptoms (e.g., weight loss) and significant reduction of yc-cytokines IL-2, IL-15 and inflammatory cytokines IFN γ 3 to 5 weeks after huPBMC transplantation via twice weekly administration of anti-NKG 2A antibodies in three representative humanized NSG mice.

Figure 10A shows the reversal of immune-mediated hair loss by BNZ-gamma in representative NSG mice. Time points are as follows: day-30 was prior to huPBMC transplantation. Day 0 was 4 weeks after transplantation of the hupmc. Day 7 is 5 weeks after huPBMC transplantation and 1 week of the BNZ- γ dosing regimen twice weekly over a two week treatment duration. Day 21 is one week after 7 weeks post-transplantation of hupmc and completion of the BNZ- γ dosing regimen twice weekly for a treatment duration of two weeks. Day 30 is just over 8 weeks after the transplantation of the hupmc and 2 weeks after completion of the BNZ- γ dosing regimen, twice weekly, over a two week treatment duration.

Figure 10B shows a comparison of serum concentrations of the circulating human inflammatory cytokines IL-6 and IFN γ in two representative NSG mice 6 weeks after huPBMC transplantation with BNZ- γ dosing regimens twice weekly, completed and not completed (PBS control) for the two weeks of treatment duration. The results were statistically significant (×), p < 0.001.

Figure 11A shows survival curves of humanized NSG mice that were started therapeutic treatment with PBS control (untreated), anti-IL-2 antibody, anti-IL-15 antibody, a combination of anti-IL-2 and anti-IL-15 antibodies, and BNZ- γ 35 days after transplantation of hupmc.

Figure 11B shows a comparison of hair regeneration levels in representative NSG mice from each of the following treatment groups after completion of a four-week treatment regimen on NSG mice 35 days after transplantation of hupmc: PBS control, anti-IL-2 Antibody (AB), anti-IL-15 AB, a combination of anti-IL-2 and anti-IL-15 AB, and BNZ-gamma.

Figure 11C shows a comparison of mean serum concentrations of circulating human inflammatory cytokines IL-6 and IFN γ from each of the following treatment groups 35 days after transplantation of hupmc after completion of a four-week treatment regimen on NSG mice: PBS control, anti-IL-2 antibody (Ab), anti-IL-15 Ab, a combination of anti-IL-2 and anti-IL-15 Ab, and BNZ-gamma.

FIG. 12 shows immunostained skin tissue from human CD8+ T cells from humanized NSG mice 3 weeks (before BNZ- γ treatment) and 7 weeks (with or without BNZ- γ treatment) after transplantation of hupPBMC. Human CD8+ T cells are highlighted with black arrows.

Figure 13A depicts the nucleotide and peptide sequences of the human CD8a chain.

Figure 13B depicts the nucleotide and peptide sequences of the human CD8 β chain.

FIG. 14 depicts the nucleotide and peptide sequences of human IL-2.

FIG. 15 depicts the nucleotide and peptide sequences of human IL-15.

Figure 16 depicts the nucleotide and peptide sequences of human NKG 2A.

Figure 17 depicts the nucleotide and peptide sequences of human NKG 2B.

Figure 18 depicts the nucleotide and peptide sequences of human NKG 2C.

Figure 19 depicts the nucleotide and peptide sequences of human NKG 2D.

Figure 20 depicts the nucleotide and peptide sequences of human NKG 2E.

Figure 21 depicts the nucleotide and peptide sequences of human NKG 2F.

Figure 22 depicts the nucleotide and peptide sequences of human NKG 2H.

Detailed description of the invention

Embodiments herein relate to compositions, methods, and kits comprising one or more therapeutic compounds that modulate signaling of at least one member of the yc-cytokine family for use in inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing autoimmune diseases (such as hair loss and hair loss-related disorders). The yc-family of cytokines includes a group of mammalian cytokines that are produced primarily by epithelial cells, stromal cells, and immune cells and control the normal and pathological activation of a wide variety of lymphocytes. Target diseases for therapeutic compounds are disclosed, as well as descriptions of methods of administration, methods of production, and methods of commercialization.

SUMMARY

More than 100 cytokines have been identified so far and are believed to be developed from the original gene bank by means of gene replication (see Bazan, j.f.1990, immunol. today 11: 350-354). To support this view, it is more common for a group of cytokines to have components in common in their multi-subunit receptor systems. The most documented common cytokine subunit in T cells is the common gamma subunit (yc subunit).

The yc subunit is common to 6 known cytokines (interleukin 2(IL-2), interleukin 4(IL-4), interleukin 7(IL-7), interleukin 9(IL-9), interleukin 15(IL-15), and interleukin 21(IL-21), collectively referred to as "yc-cytokines" or "yc family cytokines"), and plays an indispensable role in transducing cell activation signals of all of these cytokines. In addition, for each yc-cytokine, there are one or two proprietary cytokine-specific receptor subunits that, when complexed with the yc subunit, produce a fully functionalized receptor (see Rochman et al, 2009, Nat Rev immunol.9: 480-90).

The yc family of cytokines is a group of mammalian cytokines that are produced primarily by epithelial cells, stromal cells, and immune cells, and control the normal and pathological activation of a wide variety of lymphocytes. These cytokines are strictly necessary for the early development of T cells in the thymus and their homeostasis in the peripheral system. For example, T, B and NK cells in mice do not develop in the absence of the yc subunit. (see Sugamura et al, 1996, Annu. Rev. Immunol.14: 179-205).

Yc-cytokines play an important role in the development of lymphoid cells, particularly T, B and NK cells, that make up the immune system. In addition, yc-cytokines are implicated in a variety of human diseases. Thus, factors that inhibit yc-cytokine activity would provide useful tools for elucidating the developmental mechanisms of lymphocyte subpopulations and for treating immune disorders as well as yc-cytokine mediated diseases.

Germline depletion of the gene encoding the yc-subunit in mice or mutation of the yc-subunit in humans is known to cause Severe Combined Immunodeficiency (SCID) by disrupting the normal appearance or function of NK, T and B cells. In studies demonstrating that lymphocytes from these mouse and human patients lack responsiveness to yc-cytokines, the importance of the yc-subunit in the signaling of the yc-cytokine IL-2, -4, -7, -9, -15, -21 was pointed out (reviewed in Sugamura et al, 1995adv. Immunol.59: 225277). This suggests that disruption of the interaction between the yc-subunit and yc-cytokine will effectively block intracellular signaling events through the yc-cytokine family members. Thus, it is expected that antagonist peptides according to this embodiment will effectively block pathogenic changes in humans suffering from diseases mediated by misregulation of yc-cytokine family members.

Applicants propose novel compositions, methods, and kits comprising one or more therapeutic compounds that modulate signaling by at least one member of the yc-cytokine family for use in inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing autoimmune diseases (such as hair loss and hair loss-related disorders). Applicants have also designed a novel low molecular weight therapeutic compound, referred to herein as "Simul-Block," which suppresses the activity of a variety of yc-cytokines. These low molecular weight therapeutic compounds, including chemicals and peptides, are generally less immunogenic than antibodies and can be used as stand-alone methods, or complementary to antibody-mediated methods, for modulating yc-cytokine activity in clinical interventions.

Pathologies associated with yc-cytokines

Recent studies have shown that dysregulation and dysfunction of yc-cytokine expression may lead to a variety of human immunological and hematopoietic diseases.

IL-2

Although IL-2 has historically been considered the prototype T cell growth factor, the generation of knockout mice lacking IL-2 expression revealed that IL-2 is not critical for the growth or development of conventional T cells in vivo. However, overexpression of IL-2 results in preferential expansion of the T cell subset, regulatory T cells (T-regs). (see Antony et al, 2006, J.Immunol.176: 5255-66). T-regs suppress the immune response of other cells and thus act to maintain peripheral tolerance (reviewed in Sakaguchi et al, 2008, Cell 133: 775-87). Collapse of peripheral tolerance is thought to cause autoimmune disease in humans.

Thus, it is believed that the immunosuppressive function of T-regs may prevent the development of autoimmune diseases (see Sakaguchi et al, 2008, Cell 133: 775-87). T-regs are also associated with cancer, with solid tumors and hematological malignancies being associated with increased T-regs numbers (see De Rezende et al, 2010, Arch. immunological. ther. exp.58:179- > 190).

IL-4

IL-4 is a non-redundant cytokine involved in the differentiation of T helper cells to the Th2(T helper type 2) subset, which promotes the differentiation of immature B cells to IgE producing plasma cells. IgE levels are elevated in allergic asthma. Thus, IL-4 is involved in the development of allergic asthma. Antibodies targeting IL-4 may be used to treat or even prevent the onset of allergic asthma. (see Le Buanec et al, 2007, Vaccine 25: 720616).

IL-7

IL-7 is essential for B cell development and early development of T cells in the thymus. In mice, aberrant expression of IL-7 causes T cell-associated leukemia. (see Fisher et al, 1993, Leukemia 2: S66-68). However, in humans, misregulation of IL-7 does not appear to cause T cell-associated leukemia. In humans, upregulation of IL-7 alone or in combination with other yc-cytokine family member IL-15 has been associated with Large Granular Lymphocyte (LGL) leukemia.

IL-9

The considerable role of IL-9 compared to other yc-cytokine family members has not yet been characterized. Mice that have lost the IL-9 gene appear normal and do not lack any subpopulations of cells in the lymphoid and hematopoietic compartments. However, recent studies have revealed an in vivo role for IL-9 in Th17 (interleukin 17-induced T helper Cell) cytopoiesis (see Littman et al, 2010, Cell 140(6): 845-58; and Nowak et al, 2009, J.exp.Med.206: 1653-60).

IL-15

IL-15 is critically involved in the development of NK cells, NK-T cells, some subpopulations of intraepithelial lymphocytes (IEL), γ δ -T cells and memory phenotype CD 8T cells (see Waldmann,2007, J.Clin.Immunol.27: 1-18; and Tagaya et al, 1996, EMBO J.15: 4928-39). Overexpression of IL-15 in mice has led to the development of T-cell leukemias of the NK-T cell and CD8 cell types (see Fehniger et al, 2001, J.Exp.Med.193: 219-31; Sato et al, 2011 Blood, in print). The leukemias induced by these experiments appeared similar to LGL (large granular lymphocyte) leukemias in humans, as the leukemic cells expressed the CD8 antigen in both cases.

It is also suspected that the IL-15 mediated autocrine mechanism may be involved in the leukemic transformation of CD 4T lymphocytes. (see Azimi et al, 1998, Proc. Natl. Acad. Sci.95: 2452-7; Azimi et al, 1999, J.Immunol.163: 4064-72; Azimi et al, 2000, AIDS Res. hum. retroviruses 16: 1717-22; and Azimi et al, 2001, Proc. Natl. Acad. Sci.98: 14559-64). For example, CD4 tropical HTLV-I, which causes adult T cell leukemia in humans, induces autocrine growth of virally transformed T cells by producing IL-15 and IL-15 Ra (Azimi et al, 1998, Proc. Natl. Acad. Sci.95: 2452-7).

In addition to leukemia transformation, recent studies have also shown that IL-15 plays a role in the pathological development of Celiac Disease (CD), an autoimmune disease. IL-15 is known to stimulate the differentiation of NK, CD8 and intestinal intraepithelial lymphoid (IEL) cells into lymphokine-activated killer (LAK) cells by inducing the expression of cytolytic enzymes (i.e., granzyme and perforin) and interferon-gamma. Celiac disease (denoted CD from here) is an immune-mediated intestinal disease that is triggered by gluten-containing foods consumed in individuals expressing specific HLA DQ alleles.

The prevalence of the disease is 1% in the western population. The only current method of treating CD is to completely eliminate gluten in the patient's diet. The pathology of CD is mainly caused by extensive damage to the intestinal mucosa, the latter being caused by activated CD 8T cells that have penetrated the intestinal lamina propria. These CD 8T cells appear to be activated by a mechanism involving IL-15. A recent publication demonstrates that ectopic overexpression of IL-15 by intestinal cells in mice leads to the development of intestinal disease, which is very similar to the pathology in CD patients. Neutralization of IL-15 activity significantly reduces pathological changes. Thus, intervention to block IL-15 activation of CD 8T cells appears to provide an alternative strategy to conventional gluten-free diets for CD management.

IL-21

IL-21 is a recently discovered member of the yc-family. Unlike other family members, IL-21 does not appear to have a potent growth promoting effect. In contrast, IL-21 is thought to function more as a differentiation primer than as a factor controlling cell proliferation (see Tagaya,2010, J.Leuk.biol.87: 13-15).

Current strategies for treating yc-cytokine mediated disorders

Since yc-cytokines are thought to be involved in many human diseases, several approaches to the treatment of diseases involving yc-cytokines by inhibiting the activity of the yc-cytokine family have been proposed. These methods include the use of cytokine-specific monoclonal antibodies to neutralize the in vivo activity of the targeted cytokine; selectively inhibiting cytokine activity using monoclonal antibodies targeting proprietary cytokine-specific receptor subunits (subunits other than the consensus yc subunit); and the use of chemical inhibitors that block downstream intracellular cytokine signaling pathways.

Although cytokine-specific antibodies are often the first choice for designing therapeutic agents, cytokines that share a receptor component display overlapping functions (see Paul, W.E.,1989, Cell 57:521-24), and more than one cytokine may cooperate to cause disease (see the examples described herein). Thus, antibody approaches involving neutralization of a single cytokine may not always be optimal in the treatment of human diseases involving cytokines. Alternative treatment strategies may include the use of more than one antibody, wherein each antibody targets a specific cytokine involved in the pathogenesis of the disease, and/or targets a specific protein receptor involved in the pathogenesis of the disease, the activity and/or abundance of which is directly modulated by yc-cytokine signaling.

Strategies have also been proposed to design therapeutic agents that inhibit multiple cytokine functions via antibodies that recognize a common receptor component. However, the multi-subunit nature of the cytokine receptor system and the fact that functional receptors for a single cytokine may assume different configurations makes this approach difficult.

For example, a functional IL-15 receptor may be IL-15R β/γ c or IL-15R α/β/γ c. (see Dubois et al, 2002, Immunity 17: 537-47). Antibodies directed against the IL-15R β receptor (TM β 1) are potent inhibitors of IL-15 function, but only when the IL-15R α molecule is not present in the receptor complex. (see Tanaka et al, 1991, J.Immunol.147: 2222-28). Thus, the effectiveness of monoclonal antibody receptor antibodies, whether raised against a common or proprietary subunit, can be dependent on the environment and unpredictable in vivo.

The polypeptide of the therapeutic compound, fragments or other derivatives thereof, or analogs thereof, or cells expressing them, can be used as an immunogen to produce antibodies thereto. The term "immunogen" or "epitope" as used herein refers to a portion of a polypeptide that has antigenic or immunogenic activity in an animal, preferably a mammal. In preferred embodiments, the therapeutic compounds of the present invention encompass epitope-containing polypeptides as well as polynucleotides encoding such polypeptides. As used herein, an "immunogenic epitope" is defined as a portion of a protein that elicits an antibody response in an animal as determined by any method known in the art, for example, by the methods of producing antibodies described below. The term "antigenic epitope" as used herein is defined as the portion of a protein to which an antibody is capable of immunospecifically binding its antigen, as determined by any method well known in the art, e.g., by Immunoassay (Cox et al, 2004 "immunoassays methods", in Assay guide Manual [ internet ]). Immunospecific binding excludes non-specific binding, but does not necessarily exclude cross-reactivity with other antigens. The antigenic epitope need not be immunogenic. Full-length polypeptides or antigenic peptide fragments of the therapeutic compounds of the present disclosure may be used.

The software may be used by any method known in the art, such as by multiple software programs available for free, including but not limited to: bepided-2.0 (Jespersen et al, 2017Nucleic Acids Res,45: W24-W29), SVMTrip (Yao et al, 2012PLoS One,7: e45152) and ABCPred (Saha et al, 2006Proteins,65:40-8) determine epitope-bearing polypeptide regions of therapeutic compounds in the present disclosure. Antibodies are preferably prepared from these regions or from discrete fragments within these regions. However, antibodies can be prepared from any region of a peptide as described herein. Antibodies can also be developed against specific functional sites, such as ligand binding sites or sites of glycosylation, phosphorylation, myristoylation, or amidation. Peptide fragments that function as epitopes can be produced by any conventional means, such as biological production using recombinant techniques or chemically by manual or automated peptide synthesis techniques.

Various procedures known in the art can be used to produce such antibodies and fragments. The epitope-bearing polypeptides of the invention can be used to induce antibodies according to Methods well known in the art, including, but not limited to, in vivo immunization, in vitro immunization (Tomimatsu et al, 2014Methods Mol Biol,1060:297-307), and phage display Methods (Hammers et al, 2014J Invest Dermatol.,134: e 17). If in vivo immunization is used, the animal may be immunized with free peptide; however, anti-peptide antibody titers can be boosted by coupling the peptide to a macromolecular carrier such as Keyhole Limpet Hemocyanin (KLH) or tetanus toxoid. For example, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to a carrier using a more general linker such as glutaraldehyde. Antibodies raised against the polypeptides corresponding to each therapeutic compound of the invention can be obtained by direct injection of the polypeptides into an animal or by administration of the polypeptides to an animal, preferably a non-human animal. Animals such as rabbits, rats, mice and goats may be immunized with free or carrier-coupled peptides, or artificial branched forms known as Multiple Antigen Peptides (MAP), for example by intraperitoneal and/or intradermal injection of an emulsion containing about 100 μ g of the peptide or carrier protein and freund's adjuvant or any other adjuvant known to stimulate an immune response. Several booster injections may be required, for example, at about two week intervals, to provide useful anti-peptide antibody titers that can be detected, for example, by ELISA assays using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in the serum from an immunized animal can be increased by selecting anti-peptide antibodies, for example, by adsorbing to the peptide on a solid support according to methods well known in the art and eluting the selected antibodies.

For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples include hybridoma technology (Kohler et al, 1975Nature,256:495-7), trioma technology, human B-cell hybridoma technology (Kozbor et al, 1983Immunology Today,4:72-9) and EBV-hybridoma technology to produce human monoclonal antibodies (Kozbor et al, 1982Proc Natl Acad Sci,79: 6651-55). Techniques described for the production of Single chain antibody fragments (scFv) ((Et al, 2003Folia Microbiol,48:687-98) may be suitable for the generation of single chain antibodies against immunogenic polypeptides derived from therapeutic compounds of the invention.

Humanized antibodies are antibody molecules derived from antibodies of non-human species that bind a desired antigen having one or more Complementarity Determining Regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Typically, framework residues in the human framework regions will be substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction of the CDRs with framework residues to identify framework residues that are important for antigen binding and sequence, thereby identifying framework residues that are not commonly found at a particular position. (Riechmann et al, 1988Nature 332: 323-7). Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR Grafting (Williams et al, 2010 "humanizing Antibodies by CDR Grafting", in Antibody Engineering), veneering (Engineering) or resurfacing (Padlan1991Mol Immunol 28: 489-98; Studnica et al, 1994Protein Eng 7: 805-14; Roguska et al, 1994Proc Natl Acad Sci 91:969-73) and chain shuffling (Guo-Qiang et al, 2009Methods Mol Biol 562: 133-42).

Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be prepared by a variety of methods known in the art, including phage display using antibody libraries derived from human immunoglobulin sequences (Frenzel et al, 2017Transfus Med heat 44:312-18, Vaughan et al, 1996Nature 14: 309-14). Human antibodies that recognize selected epitopes can also be produced using a technique known as "guided selection". In this method, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of fully human antibodies that recognize the same epitope. (Jespers et al, 1994Biotechnology 12: 899-903).

In addition, transgenic mice can be used to express human antibodies against immunogenic polypeptides derived from the therapeutic compounds of the invention (Laffleur et al, 2012Methods Mol Biol,901: 149-59). Transgenic mice that are incapable of expressing functional endogenous immunoglobulins can be used to express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, in addition to human heavy and light chain genes, human variable, constant and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can become non-functional when introduced into the human immunoglobulin locus by homologous recombination, separately or simultaneously. Specifically, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells were expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to produce homozygous progeny that express the human antibody. The transgenic mice are immunized in the normal manner with a selected antigen, e.g., all or a portion of a polypeptide corresponding to a therapeutic compound of the invention. Monoclonal antibodies directed against the antigen can be obtained from immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation, subsequently undergo class switching and somatic mutation. Thus, using this technique, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced. For a summary of this technique for the production of human antibodies, see Lonberg et al, 1995Int Rev Immunol.13: 65-93.

Antibodies of the invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab') fragments, fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies directed against an antibody of the invention), and epitope-binding fragments of any of the above. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen. The immunoglobulin molecules of the invention may be of any type (e.g., IgG, IgE, IgM, Ig | D, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. In a preferred embodiment, the immunoglobulin is an IgG1 isotype. In another preferred embodiment, the immunoglobulin is an IgG2 isotype. In another preferred embodiment, the immunoglobulin is an IgG4 isotype. The immunoglobulin may have a heavy chain and a light chain. A series of IgG, IgE, IgM, IgD, IgA and IgY heavy chains can be paired with light chains in either the kappa or lambda forms.

Targeting JAK3, an alternative example of the existence for inhibition of multiple yc-cytokines

The interaction between the yc-subunit and yc-cytokines results in the activation of an intracellular protein tyrosine kinase known as Janus kinase 3(Jak 3). In turn, Jak3 phosphorylates a variety of signaling molecules, including STAT5 and PI3 kinases. The interaction of the yc-subunit with Jak3 is very specific. In fact, no other receptor molecules recruit Jak3 for signaling. (see O' Shea,2004, Ann. Rheum. Dis.63 (suppl. II): ii 67-7). Thus, inhibition of cytokine signaling by the yc-subunit can be achieved by blocking the activity of Jak3 kinase. Thus, a variety of small molecule chemical inhibitors targeting the kinase activity of Jak3 have been introduced to the market. (see Pesu et al, 2008, Immunol. Rev.223: 132-142). One such example is CP690,550.

The major drawback of these protein kinase inhibitors is the lack of specificity for Jak3 kinase. These drugs block the binding of ATP (adenosine triphosphate) molecules to Jak3 kinase, a common biochemical reaction of many protein kinases, and thus tend to block the action of a variety of intracellular protein kinases not related to Jak3 kinase, whose action is strictly essential for the health of normal cells in a variety of tissues. Thus, there is a need for more specific signaling inhibitors through the yc-subunit.

Therefore, alternative non-small molecule chemical strategies for treating diseases involving yc-cytokines are highly desirable.

Discovery of the gammac-cassette

The C-terminus (D-helix) of yc-cytokines contains proposed sites for interaction with the common yc-subunit of multiple unit cytokine receptors (Bernard et al, 2004J.biol.chem.279: 24313-21). Comparison of the biochemical properties of the amino acids of all yc-cytokines identified in mice and humans reveals that the chemical properties of the amino acids at many positions in the D-helix of members of the yc-cytokine family, such as hydrophobicity, hydrophilicity, basicity/acidity, are conserved (if not identical).

In contrast, the sequence of IL-13, which is associated with the yc-cytokine IL-4 but does not bind to the yc-subunit, does not show significant homology to the yc-cytokine in the D-helix region, indicating that sequence homology in the D-helix region is associated with the binding of the yc-subunit. As shown in fig. 1A, alignment of the amino acid sequences of the D-helical regions of the yc-cytokine family members in humans reveals motifs of intermediate sequence homology among these cytokines, which are referred to herein as "yc-boxes".

The yc-box (SEQ ID NO:9) contains 19 amino acids, where in 19 positions 4, 5 and 13 are fully conserved, phenylalanine, leucine and glutamine, respectively. Less conservation is observed at positions 6, 7 and 11 of the yc-box, where the amino acid is one of two or three related amino acids sharing physicochemical properties: position 6 may be occupied by the polar amino acids glutamic acid, asparagine or glutamine; the nonpolar amino acids serine or arginine may occupy position 7; and position 11 may be occupied by one of the nonpolar aliphatic amino acids leucine or isoleucine. Positions 9 and 16 may be occupied by the nonpolar amino acid isoleucine or the polar amino acid lysine. See fig. 1B. In the subfamily of yc-cytokines, some differences in the amino acid composition of the yc-box were observed at positions 9 and 16. Comparison of yc-cytokines between species indicates that isoleucine is usually present at positions 9 and 16in the IL-2/15 subfamily, while other yc-family members usually have lysine in these positions. Without wishing to be bound by a particular theory, isoleucine and lysine are biochemically distinct and thus may confer a specific conformational difference between the IL-2/15 subfamily and other yc-cytokines.

The conservation of the yc-box motif between yc-cytokines is supported by the following findings: glutamine (Gln, Q) residues located in the D-helical region are critical for the binding of yc-cytokines to yc-subunits (Bernard et al, 2004J.biol. chem.279: 2431321).

Modulators of yc-cytokine activity

The activity of yc-family cytokines can be blocked by disrupting the interaction between the yc-cytokine and the yc-subunit, for example by introducing competitive inhibitors that can interact with the yc-subunit without stimulating signaling through the multi-subunit cytokine receptor. Without being bound by a particular theory, the conserved yc-box motif involved in the binding of yc-family cytokines to the yc-subunit presents a central basic amino acid sequence that can be used to design peptide modulators of yc-cytokine signaling.

The core yc-box amino acid sequence comprises: D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO:2) (wherein X represents any amino acid). Embodiments described herein relate to tailored peptide derivatives of core yc-box amino acid sequences that are capable of modulating the activity of one or more yc-cytokines. Tailored peptide derivatives include any peptide whose partial amino acid sequence exhibits about 50%, 50% -60%, 60% -70%, 70% -80%, 90%, 95%, 97%, 98%, 99% or 99.8% identity to the core yc-box amino acid sequence. Tailored peptide derivatives also include any peptide wherein a portion of the amino acid sequence of the peptide derivative comprises amino acids having similar physicochemical properties as the amino acids of the core yc-box. For example, amino acids with similar physicochemical properties would include phenylalanine, tyrosine, tryptophan, and histidine, which are aromatic amino acids. Figure 2 shows a schematic representation of amino acids with similar physicochemical properties that can be substituted for amino acids comprising the core yc-box. The peptide derivative of the core yc-cassette may be 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25-30, 30-35, 35-40, 40-45, 45-50 or more than 50 amino acids in length. In some embodiments, the custom-made peptide derivatives may be conjugated to N-terminal, C-terminal, and/or side chain residues of existing biological proteins/peptides.

Based on the identification of conserved yc-box motifs in yc-subunit binding cytokines, applicants designed novel 19-mer custom-derived peptides that combine human IL-2 and IL-15 yc-An artificial composite peptide of the amino acid sequence of the cassette. The 19-mer peptide, referred to herein as BNZ- γ, consists of the following amino acid sequence: I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1), wherein the amino acids shown by bold characters are conserved between IL-2 and IL-15, and underlined amino acids represent positions wherein the physicochemical properties of the amino acids are conserved.

Applicants found that 19-mer BNZ- γ suppressed IL-15 and IL-9 induced cell proliferation, but not IL-3 or IL-4 induced cell proliferation. See figure 3A and example 2. Applicants have also demonstrated that BNZ-gamma inhibits the IL-15 mediated phosphorylation of the cytokine signaling molecule STAT-5 in cells. See figure 4 and example 5. These results demonstrate that tailored peptide derivatives of conserved yc-box motifs are capable of modulating the activity of a variety of yc-cytokines.

Several embodiments relate to one or more therapeutic compounds that modulate signaling of at least one member of the yc-cytokine family for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing autoimmune diseases (e.g., hair loss and hair loss-related disorders). In some embodiments, the therapeutic compound is one or more of a yc-cytokine antagonist peptide, a yc-cytokine antagonist peptide derivative, an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, or a combination thereof.

In some embodiments, yc-cytokine antagonistic peptides and derivatives thereof, also referred to herein as custom-derived peptides or complex peptide derivatives of the 19-mer BNZ-gamma amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1), can inhibit the activity of one or more yc-cytokines. Customized peptide derivatives of the 19-mer BNZ-gamma amino acid sequence include any peptide, a portion of which exhibits about 50%, 50% -60%, 60% -70%, 70% -80%, 90%, 95%, 97%, 98%, 99% or 99.8% identity to the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1). Customized peptide derivatives also include any peptide wherein a portion of the amino acid sequence of the peptide derivative comprises an amino acid with physicochemical properties similar to the amino acid of the sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-N-T-S (SEQ ID NO: 1).

In several embodiments, the amino acid residues of the custom-derived peptides retain physicochemical properties similar to those of BNZ- γ, but exhibit different biostatic specificities for 6 yc-cytokine family members from those of the original 19-mer peptides. Peptide derivatives of BNZ- γ can be 19, 20, 21, 22, 23, 24, 25-30, 30-35, 35-40, 40-45, 45-50, or more than 50 amino acids in length.

In some embodiments, the custom peptide derivatives may be conjugated to the N-terminal, C-terminal, and/or side chain residues of existing biological proteins/peptides. In some embodiments, the peptide derivative of BNZ- γ can be conjugated to other moieties through the N-terminus, C-terminus, or side chain of the composite peptide. Other moieties may include proteins or peptides that stabilize the composite peptide or other moieties, including but not limited to: bovine Serum Albumin (BSA), albumin, Keyhole Limpet Hemocyanin (KLH), Fc region of IgG, biological protein functioning as a scaffold, antibody against cell-specific antigen, receptor, ligand, metal ion, and polyethylene glycol (PEG).

In some embodiments, any of the tailored peptide derivatives disclosed herein may comprise one or more intra-peptide hydrocarbon linker elements. In some embodiments, 19-mer BNZ- γ (SEQ ID NO:1) comprises one or more intrapeptide hydrocarbon linker elements. In some embodiments, 19-mer BNZ- γ (SEQ ID NO:1) comprises one or more intrapeptide hydrocarbon linker elements that link two separate amino acids positioned 4 residues apart on SEQ ID NO: 1. In some embodiments, 19-mer BNZ- γ (SEQ ID NO:1) comprises one or more intrapeptide hydrocarbon linker elements that link two separate amino acids positioned 7 residues apart on SEQ ID NO: 1. In some embodiments, 19-mer BNZ- γ (SEQ ID NO:1) comprises one or more intrapeptide hydrocarbon linker elements connecting two separate amino acids that are positioned 4 residues apart from one another at positions on SEQ ID NO:1 and 7 residues apart from one another at positions on SEQ ID NO: 1.

Several embodiments relate to a custom-derived peptide of amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-N-T-S (SEQ ID NO:1) that can inhibit the activity of one or more yc-cytokines. Customized peptide derivatives of amino acid sequences include any peptide, a portion of which shows about 50%, 50% -60%, 60% -70%, 70% -80%, 90%, 95%, 97%, 98%, 99% or 99.8% identity to the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1). Customized peptide derivatives also include any peptide wherein a portion of the amino acid sequence of the peptide derivative comprises an amino acid with physicochemical properties similar to the amino acid of the sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-N-T-S (SEQ ID NO: 1).

In several embodiments, the amino acid residues of the custom-derived peptides retain physicochemical properties similar to those of SEQ ID No. 1, but exhibit different biostatic specificities for the 6 yc-cytokine family members from those of the original 19-mer peptides. The peptide derivative of SEQ ID NO. 1 may be less than 19, 20, 21, 22, 23, 24, 25-30, 30-35, 35-40, 40-45, 45-50 or more than 50 amino acids in length.

In some embodiments, the custom peptide derivatives may be conjugated to the N-terminal, C-terminal, and/or side chain residues of existing biological proteins/peptides. In some embodiments, the composite peptide of SEQ ID NO:1 may be conjugated to other moieties through the N-terminus, C-terminus, or side chain of the composite peptide. In some embodiments, the additional moiety may include proteins or peptides that stabilize the composite peptide or additional moiety, including but not limited to: bovine Serum Albumin (BSA), albumin, Keyhole Limpet Hemocyanin (KLH), Fc region of IgG, biological protein functioning as a scaffold, antibody against cell-specific antigen, receptor, ligand, metal ion, and polyethylene glycol (PEG).

In some embodiments, any of the tailored peptide derivatives disclosed herein may comprise one or more intra-peptide hydrocarbon linker elements. In some embodiments, the composite peptide of SEQ ID NO:1 comprises one or more intrapeptide hydrocarbon linker elements. In some embodiments, the composite peptide of SEQ ID No. 1 comprises one or more intrapeptide hydrocarbon linker elements that link two separate amino acids that are positioned 4 residues apart on SEQ ID No. 1. In some embodiments, the composite peptide of SEQ ID No. 1 comprises one or more intrapeptide hydrocarbon linker elements that link two separate amino acids that are located 7 residues apart on SEQ ID No. 1. In some embodiments, the composite peptide of SEQ ID No. 1 comprises one or more intrapeptide hydrocarbon linker elements that link two separate amino acids that are positioned 4 residues apart from each other at position 1 and 7 residues apart from each other at position 1.

Several embodiments relate to tailored peptide derivatives of the yc-box motif of IL-15, IL-2, IL-21, IL-4, IL-9, or IL-7, depicted in FIG. 1A. Other embodiments relate to custom-derived peptides that are artificial composite peptides that combine amino acid sequences of two or more of human IL-15, IL-2, IL-21, IL-4, IL-9, and IL-7 yc-box motifs. Several embodiments relate to tailored peptide derivatives of a yc-box motif of IL-15, IL-2, IL-21, IL-4, IL-9, or IL-7 having a partial amino acid sequence that exhibits about 50%, 50% -60%, 60% -70%, 70% -80%, 90%, 95%, 97%, 98%, 99%, or 99.8% identity to the amino acid sequence of the yc-box motif of IL-15, IL-2, IL-21, IL-4, IL-9, or IL-7. Tailored peptide derivatives of the yc-box motif of IL-15, IL-2, IL-21, IL-4, IL-9 or IL-7 also include any peptide in which a portion of the amino acid sequence of the peptide derivative comprises amino acids having similar physicochemical properties as the amino acids of the yc-box motif sequence of IL-15, IL-2, IL-21, IL-4, IL-9 or IL-7.

Several embodiments relate to tailored peptide derivatives that will inhibit the function of one, all, or a selective member of the yc-cytokines. In some embodiments, the tailored peptide derivative selectively targets individual yc-cytokine family members. For example, the tailored peptide derivative is capable of selectively inhibiting the function of IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21. In other embodiments, the tailored peptide derivative is capable of inhibiting 2 or more yc-cytokine family members.

For example, the tailored peptide derivatives of this embodiment are capable of selectively inhibiting the function of: IL-2 in combination with one or more of IL-4, IL-7, IL-9, IL-15, and IL-21; IL-4 in combination with one or more of IL-2, IL-7, IL-9, IL-15, and IL-21; IL-7 in combination with one or more of IL-2, IL-4, IL-9, IL-15, and IL-21; IL-9 in combination with one or more of IL-2, IL-4, IL-7, IL-15, and IL-21; IL-15 in combination with one or more of IL-2, IL-4, IL-7, IL-9, and IL-21; or IL-21 in combination with one or more of IL-2, IL-4, IL-7, IL-9, and IL-15. In other embodiments, the tailored peptide derivatives are capable of targeting all yc-cytokine family members globally.

Without wishing to be bound by a particular theory, the tailored peptide derivatives are capable of inhibiting the function of all or selected members of the yc-cytokines by reducing the binding of the yc-cytokine to the yc-subunit, e.g., as competitive inhibitors. Such tailored peptide derivatives may be used in different applications, including as clinical drugs.

Several embodiments relate to tailored peptide derivatives that will modulate (including enhance or decrease) the function of one, two or more selective members of the yc-cytokine. In some embodiments, the tailored peptide derivative selectively targets individual yc-cytokine family members. For example, the tailored peptide derivatives can selectively enhance or inhibit the function of IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21. In other embodiments, the tailored peptide derivative is capable of enhancing or inhibiting two or more yc-cytokine family members.

In some embodiments, one or more of the tailored peptide derivatives of the conserved yc-box motifs disclosed herein are capable of inhibiting the activity of one or more yc-cytokines. In some embodiments, one or more of the tailored peptide derivatives of the conserved yc-box motifs disclosed herein are capable of inhibiting the activity of one or more yc-cytokines by inhibiting cell proliferation induced by the one or more yc-cytokines. In some embodiments, one or more of the tailored peptide derivatives of the conserved yc-box motifs disclosed herein are capable of inhibiting the activity of one or more yc-cytokines by inhibiting the phosphorylation of intracellular cytokine signaling molecules mediated by the one or more yc-cytokines. In some embodiments, one or more of the tailored peptide derivatives of the conserved yc-box motifs disclosed herein are capable of inhibiting the activity of one or more yc-cytokines by inhibiting cell proliferation induced by the one or more yc-cytokines, and by inhibiting phosphorylation of intracellular cytokine signaling molecules mediated by the one or more yc-cytokines. In some embodiments, one or more of the tailored peptide derivatives of the conserved yc-box motifs disclosed herein are capable of inhibiting the activity of one or more yc-cytokines through one or more other mechanisms.

In some embodiments, one or more of the peptide sequences disclosed herein inhibit proliferation of one or more cell types induced by one or more of the cytokines disclosed herein (e.g., IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). In some embodiments, one or more of the peptide sequences disclosed herein inhibit proliferation of one or more cell types induced by all of the cytokines disclosed herein. In some embodiments, one or more of the peptide sequences disclosed herein inhibit proliferation of one or more cell types induced by some, but not all, of the cytokines disclosed herein. In some embodiments, SEQ ID NO 1 inhibits IL-2, IL-9, and IL-15 induced cell proliferation.

In some embodiments, one or more of the tailored peptide derivatives of the conserved yc-box motifs disclosed herein are capable of inhibiting the activity of one or more yc-cytokines by inhibiting the phosphorylation of one or more intracellular cytokine signaling molecules mediated by one or more yc-cytokines disclosed herein (e.g., IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). In some embodiments, one or more of the tailored peptide derivatives of the conserved yc-box motifs disclosed herein are capable of inhibiting phosphorylation of one or more intracellular cytokine signaling molecules mediated by all yc-cytokines disclosed herein. In some embodiments, one or more of the tailored peptide derivatives of the conserved yc-box motifs disclosed herein are capable of inhibiting phosphorylation of one or more intracellular cytokine signaling molecules mediated by some, but not all, yc-cytokines disclosed herein.

In addition, for example, the peptides disclosed herein can be used to inhibit IL-15 mediated phosphorylation of the intracellular cytokine signaling molecule STAT-5.

Provided herein are composite peptides and compositions, methods and kits that modulate yc-cytokine signaling. The terms "composite peptide", "composite peptide derivative", "tailored peptide", "antagonist peptide derivative", "oligopeptide", "polypeptide", "peptide" and "protein" are used interchangeably in reference to the "tailored peptide derivative" provided according to the present embodiment and may be used to designate a series of amino acid residues of any length. The peptides of this embodiment may be linear or cyclic. The peptide of the present embodiment may include a natural amino acid, an unnatural amino acid, (D) an amino acid in stereochemical configuration, (L) an amino acid in stereochemical configuration, (R) an amino acid in stereochemical configuration, (S) an amino acid in stereochemical configuration, or a combination thereof.

The peptides of this embodiment may also comprise one or more rare amino acids (such as 4 hydroxyprolines or hydroxylysines), organic acids or amides, and/or derivatives of common amino acids, such as C-terminal carboxylates with esterification (e.g., benzyl, methyl, or ethyl ester) or amidation, and/or amino acids with N-terminal amino modifications (e.g., acetylated or alkoxycarbonylamino), with or without any of a variety of side chain modifications and/or substitutions. Side chain modifications, substitutions, or combinations thereof that may be present in the tailored peptide derivatives of the present embodiments include, but are not limited to: alpha-methyl, alpha-alkenyl, alkylation, methylation, benzylation, tert-butylation, tosylation, alkoxycarbonylamino and the like.

Residues other than the common amino acids that may be present include, but are not limited to: penicillamine, tetramethylene cysteine, pentamethylene cysteine, mercaptopropionic acid, norleucine, pentamethylene-mercaptopropionic acid, 2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine, aminoisobutyric acid, diaminobutyric acid, aminoadipic acid, aminomethylbenzoic acid, and diaminopropionic acid.

The peptides of the present embodiment can be produced and obtained by various methods known to those skilled in the art. For example, the peptide may be produced by genetic engineering, production of the peptide based on a nucleotide sequence encoding the peptide of the embodiment, or chemical synthesis of the peptide by peptide solid phase synthesis or the like, or a combination thereof. Natural or unnatural amino acids in (D) and (L) or (R) and (S) stereochemical configurations can be readily incorporated by those skilled in the art of solid phase peptide synthesis. It will also be apparent to those skilled in the art of solid phase peptide synthesis that peptides comprising one or more of the intrapeptide hydrocarbon linker elements of the present embodiments are produced and obtained using alpha-substituted (e.g., alpha-alkenyl) natural or unnatural amino acids in one or more of (D), (L), (R), or (S) stereochemical configurations, or combinations thereof. In some embodiments, an intrapeptide hydrocarbon linker element linking an alpha-substituted amino acid (e.g., an alpha-alkenyl amino acid) can be produced by catalyzing one or more ring closing metathesis reactions. In some embodiments, one or more intrapeptide hydrocarbon linker elements can be generated by using benzylidene bis (tricyclohexyl-phosphine) -dichlororuthenium (Grubb's catalyst) to catalyze a ring closing metathesis reaction on a resin-bound peptide during peptide synthesis.

The peptides of this embodiment may also comprise two or more alpha-alkenyl substituted amino acids. In some embodiments, the two or more α -alkenyl substituted amino acids are linked via one or more intrapeptide hydrocarbon linker elements incorporated at the α -alkenyl substituted amino acids. In some embodiments, the α -alkenyl substituted amino acids are used to catalyze the formation of hydrocarbon linker elements within peptides through ring closing metathesis reactions during peptide synthesis. The intra-peptide linker element links individual amino acids on the same sequence of the custom-made peptide derivatives of the present disclosure. In some embodiments, the peptides of the present disclosure are linear or cyclic.

In some embodiments, one or more intrapeptide hydrocarbon linker elements are incorporated at amino acid positions that are correlated with single alpha-helical turns in the secondary structure of the composite peptide. In some embodiments, when the composite peptide comprises one or more non-contiguous single alpha-helical turns, the amino acid positions associated with the single alpha-helical turn of the composite peptide correspond to amino acid positions i and i +4 of the composite peptide, wherein i is the first amino acid position of the single alpha-helical turn and i +4 is the last amino acid position of the single alpha-helical turn, and wherein amino acid positions i and i +4 comprise alpha-alkenyl substituted amino acids, and wherein the positions of i and i +4 are 4 residues apart (4 apart).

In some embodiments, one skilled in the art of solid phase peptide synthesis can readily synthesize a composite peptide comprising more than one intra-peptide hydrocarbon linker element such that the composite peptide comprises more than one single alpha-spiro turn. In some embodiments, more than one single alpha-helical turn is non-contiguous, i.e., more than one single alpha-helical turn does not share substituted amino acids. For example, in some embodiments, a composite peptide may comprise one or more intrapeptide hydrocarbon linker elements of formula 1 (see table 1) that span more than one non-contiguous single alpha-helical turn of the composite peptide.

Without wishing to be bound by any particular peptide comprising one or more intrapeptide hydrocarbon linker elements of this embodiment, a generic peptide example comprising an intrapeptide hydrocarbon linker element linking two separate one amino acids at positions 4 residues apart or one alpha-helical rotation angle (positions i and i +4) may have S-pentenylalanine (S5Ala) incorporated at each position i and i +4 during solid phase synthesis of the peptide while the peptide is still resin bound (resin bound) on the solid support prior to catalysis of ring closing metathesis using a grubbs catalyst. This will result in a peptide sequence comprising the intra-peptide hydrocarbon linker element (SEQ ID NO:23) depicted below positioned 4 residues apart:

in some embodiments, one or more intrapeptide hydrocarbon linker elements are incorporated at amino acid positions that are correlated with double alpha-helical turns in the secondary structure of the composite peptide. In some embodiments, when the composite peptide comprises one or more non-contiguous double alpha-helical turns, the amino acid positions associated with the double alpha-helical turns of the composite peptide correspond to amino acid positions i and i +7 of the composite peptide, wherein i is the first amino acid position of the double alpha-helical turn and i +7 is the last amino acid position of the double alpha-helical turn, and wherein amino acid positions i and i +7 comprise alpha-alkenyl substituted amino acids, and wherein the positions of i and i +7 are 7 residues apart (separated by 7).

Without wishing to be bound by any particular peptide comprising one or more intrapeptide hydrocarbon linker elements of this embodiment, one intrapeptide hydrocarbon linker element comprising two separate amino acids at positions 7 residues apart, or a general example of a peptide of two alpha-helical rotation angles (positions i and i +7), may have R-octenylalanine (R8Ala) incorporated at position i and S-pentenylalanine (S5Ala) incorporated at position i +7 during solid phase synthesis of the peptide while the peptide is still resin bound to the solid support prior to catalysis of ring closing metathesis using a grubbs catalyst. This will result in a peptide sequence comprising the intra-peptide hydrocarbon linker elements depicted below (SEQ ID NO:24) positions 7 residues apart:

in some embodiments, a composite peptide comprising more than one intra-peptide hydrocarbon linker element such that the composite peptide comprises more than one double alpha-helical turn can be readily synthesized by one skilled in the art of solid phase peptide synthesis. In some embodiments, more than one double α -helix turn is non-continuous, i.e., more than one double α -helix turn does not share substituted amino acids. For example, in some embodiments, a composite peptide may comprise one or more intrapeptide hydrocarbon linker elements of formula 2 spanning more than one non-contiguous double α -helical turn of the composite peptide (see table 1).

One skilled in the art of solid phase peptide synthesis can readily synthesize the peptides of this embodiment comprising more than one intra-peptide hydrocarbon linker element by incorporating alpha-alkenyl substituted amino acids at pairs of non-overlapping amino acid positions of the peptide during solid phase peptide synthesis while the peptide is still resin bound to a solid support, prior to catalyzing a ring closing metathesis reaction using a grubbs catalyst, wherein the position of each alpha-alkenyl substituted amino acid in the pair of amino acids is separated from a single alpha-spiro turn (4 residues apart) or a double alpha-spiro turn (7 residues apart). In some embodiments, a single peptide may comprise more than one intra-peptide hydrocarbon linker element spanning a single alpha-spiro turn (separated by 4 residues), may comprise hydrocarbon linker elements spanning a double alpha-spiro turn (separated by 7 residues), or may comprise a combination of single alpha-spiro turn (separated by 4 residues) and double alpha-spiro turn (separated by 7 residues) intra-peptide hydrocarbon linker elements.

Peptides comprising one or more intra-peptide hydrocarbon linker elements of the present embodiment can be made by solid phase peptide synthesis using commercially available Boc or Fmoc protected alpha-alkenyl substituted amino acids of either the natural or non-natural (D) and (L) or (R) and (S) stereochemical configurations. Fmoc-protected α -alkenyl substituted amino acids and resulting hydrocarbon linker elements after ring closing metathesis reactions useful for synthesizing the custom peptide derivatives of the present embodiments include, but are not limited to, table 1:

TABLE 1

In some embodiments, the hydrocarbon linker within the peptide may be further functionalized by one or more chemical reactions. In some embodiments, one or more carbon-carbon double bonds present in a hydrocarbon linker within a peptide (e.g., formula 1-formula 2 in table 1) may be used in organic chemical reactions to add one or more additional chemical functional groups. For example, an alkene reaction may be used to tailor peptide derivatives comprising one or more intrapeptide hydrocarbon linker elements of the present embodiments. Non-limiting examples of olefin reactions include hydroboration, hydromercurization, hydration, chlorination, bromination, addition of HF, HBr, HCl or HI, dihydroxylation, epoxidation, hydrogenation, and cyclopropanation. In some embodiments, one or more additional chemical functional groups of the intra-peptide hydrocarbon linker element may be obtained after the olefin reaction. Non-limiting examples include the covalent addition of one or more chemical group substituents, such as nucleophilic reactions with epoxy and hydroxyl groups, and the like. In some embodiments, an olefinic reaction may be used to attach biotin, radioisotopes, therapeutic agents (non-limiting examples include rapamycin, vinblastine, paclitaxel, etc.), non-protein fluorescent chemical groups (non-limiting examples include FITC, hydrazide, rhodamine, maleimide, etc.), and protein fluorescent groups (non-limiting examples include GFP, YFP, mCherry, etc.) to one or more inter-and/or intra-peptide hydrocarbon linker elements of the present embodiments.

Non-limiting examples of composite peptides comprising one or more intra-peptide hydrocarbon linker elements are provided in table 2. The examples in table 2 are not limited to any particular alpha-alkenyl substituted amino acids in the tailored peptide derivatives of the present embodiments that may be used to synthesize the single alpha-spiro turn (4 apart) of the present embodiments, and/or the double alpha-spiro turn (7 apart) of the hydrocarbon linker element within the peptide, and/or any particular stereochemical configuration of the amino acid (e.g., (D) stereochemical configuration denoted as "D" in table 2).

TABLE 2

Subscript denotes the corresponding pair of hydrocarbon-linked alpha-alkenyl substituted amino acids

In some embodiments, the therapeutic compound may be an antibody. Antibodies can be developed to target yc-cytokines, such as IL-2 or IL-15, or to target specific protein receptors whose activity and/or abundance is directly regulated by cytokine signaling, such as proteins of the transmembrane glycoproteins CD8 or NKG 2C-type lectin receptor family, both of which are expressed on T-lymphocytes.

Some embodiments also relate to polynucleotides comprising nucleotide sequences encoding the peptides and antibodies of the invention. "nucleotide sequence", "polynucleotide" or "nucleic acid" are used interchangeably and are understood to mean double-stranded DNA, single-stranded DNA or a transcription product of said DNA (e.g., an RNA molecule). The polynucleotide may be administered to and expressed by a cell or subject, rather than the peptide itself. Several embodiments also relate to genetic constructs comprising polynucleotide sequences encoding the peptides of the invention. The genetic construct may also contain additional regulatory elements, such as promoters and enhancers, and optionally selectable markers.

Methods of treating yc-cytokine mediated diseases

Several embodiments relate to the use of therapeutic compounds such as yc-antagonistic peptides, cytokine targeting antibodies, and/or antibodies targeting specific protein receptors whose activity and/or abundance is directly modulated by cytokine signaling in the treatment of yc-cytokine mediated diseases. The use of therapeutic compounds according to this embodiment allows for flexibility in design and combination, which achieves more comprehensive results not achievable with conventional strategies using small molecule chemical inhibitors or anti-cytokine receptor antibodies.

Described herein are novel methods of modulating the effects of yc-family cytokines. This procedure can lead to effective clinical intervention for the treatment of autoimmune diseases such as alopecia and alopecia-related disorders.

In some embodiments, compositions, methods, and kits for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing at least one hair loss-related disorder are described. In some embodiments, the therapeutic compounds described herein may be used to inhibit, ameliorate, reduce, treat, delay the onset of, or prevent one or more of the following diseases: alopecia areata, alopecia totalis, sub-alopecia totalis (alpoteca subtotalis), alopecia universalis, diffuse alopecia (alpoteca diffusa), alopecia areata (ophheas-type alpoteca areata), and other immune-mediated diseases associated with alopecia, such as lichen planus, lichen sclerosus, lichen atrophicus, atopy, atopic dermatitis, psoriasis vulgaris, psoriasis capitis (psioasis capitis), trichomonas psoriasis (psis guttate), psoriasis parapropathia (psioasis invers), psoriatic arthritis, eczema, pemphigus vulgaris, pemphigus proliferatum, pemphigoid, mucocutaneous pemphigoid, cicatricial mucocutaneous pemphigoid, thyroid disorders, hypothyroidism, urticaria, local and other immune-mediated diseases associated with alopecia areata, such as, Prurigo, rosacea vitiligo, vitiligo and graft-versus-host disease (GvHD).

Several embodiments relate to therapeutic compounds that will modulate the signaling of all or selective members of the yc-cytokine. In some embodiments, the therapeutic compound selectively modulates signaling by individual yc-cytokine family members. In other embodiments, the therapeutic compound may globally modulate the signaling of all yc-cytokine family members (Simul-Block). In some embodiments, the therapeutic compound can selectively modulate signaling of a subpopulation of yc-cytokines. Without wishing to be bound by a particular theory, the therapeutic compound may modulate the function of all or selective members of the yc-cytokine by reducing its binding to the yc-subunit, e.g., as a competitive inhibitor, or by modulating the activity and/or abundance of a particular protein receptor that is itself directly regulated by yc-cytokine signaling.

Several members of the yc-cytokine family have been implicated in alopecia disease progression. Alopecia is an immune-mediated skin disorder in which there is a T cell hyperproliferative environment that supports the targeting of T cells to the hair follicle autoantigens that ultimately lead to alopecia. IL-2 and IL-15 expression was elevated in focal scalp biopsies of patients (Fuentes-Ducuman et al, 2016 Exp Dermatol 4:282-6, Suarez-Farinas et al, 2015J. allergy Clin. Immunol.136:1277-87, Waldmann 2013J Investig Dermatol Symp Proc16: S28-30), and antibodies targeting γ c-cytokines IL-2 and IL-15 each showed inhibitory activity in a mouse model of hair loss, but none of the blocking antibodies alone could reverse the established condition Xing et al, 2014Nat Med 9: 1043-9). Expression of IL-21 was elevated in serum of baldness patients compared to healthy controls (Atwa et al, 2016Int J Dermatol 55:666-72), and genome-wide association studies also had IL-2 and IL-21 positively correlated with baldness (Jagilska et al, 2012J Invest Dermatol 132:2192-7, Petukhova et al, 2010 Nature466: 113-7).

Vitiligo is an immune-mediated skin disorder associated with the influx of T cells in the epidermis, which results in melanocyte destruction and white spots on body surfaces. Recent studies have shown that blocking IL-15 signaling via antibody therapy is an effective therapeutic strategy in mice with established vitiligo (Richmond et al, 2018Sci Transl Med 10: 450). Interestingly, the antibodies used in the studies targeted CD122, a common proprietary cytokine-specific receptor subunit of IL-15 and IL-2. In fact, IL-2 expression has been shown to be elevated in the serum of patients with localized and systemic vitiligo, and is positively correlated with disease severity (Sushama et al, 2018J Cosmet Dermatol 00: 1-5).

Pemphigoid and pemphigus are immune-mediated skin conditions characterized by the presence of large, liquid-filled blisters on the body surface. In early studies, both pemphigoid and pemphigus fluid from human patients showed elevated IL-2 activity (Grando et al, 1989, Arch Dermatol.125: 925-30). Patients with pemphigoid also show increased T-cell activation and elevated IL-2 levels (Schaller et al, 1990, Arch Dermatol. Res.282: 223-6). Separate studies evaluated IL-15 levels in pemphigoid and pemphigus patients, and found that patients with either disease exhibited increased IL-15 serum levels that were positively correlated with disease severity (D' Auria et al, 1999, Arch Dermatol. Res.291: 354-6).

Certain yc-cytokines have been shown to be positively associated with psoriasis. Psoriasis is an immune-mediated skin condition characterized by squamous red patches of extra skin cells, which are usually dry, itchy, and sometimes painful. Expression of IL-15 is elevated in skin lesions in patients with psoriasis (Waldmann 2013J Investig Dermatol Symp Proc16: S28-30). IL-15 specific antibodies, which effectively interfere with the assembly of the IL-15 cytokine-receptor signaling complex, reduce the severity of disease in a human psoriasis xenograft model (Villadsen et al, 2003, J.Clin.invest.112: 1571-80). Another yc-cytokine, IL-21, has also been shown to be elevated in psoriasis patients and to be positively correlated with disease severity (Caruso et al, 2009Cell Cycle 8:3629-30, Botti et al, 2012Curr Pharm Biotechnol 13:1861-7, He et al, 2012, br.j. dermotol.167: 191-3). Blocking cytokines via anti-IL-21 antibody treatment resulted in a significant reduction in keratinocyte proliferation and inflammation in a human psoriasis xenograft mouse model (Caruso et al, 2009nat. med.15: 1013-5).

Graft versus host disease (GvHD) can often arise after hematopoietic cell transplantation in patients because host cells are recognized as foreign entities by donor T lymphocytes. GvHD manifests itself as host organ tissue damage because donor-derived T cells differentiate into CD4 and CD8 effector cells, producing pro-inflammatory cytokines and a direct CD 8T cell cytotoxic effect. Members of the yc-cytokine family are known to be involved in the activation of CD4 and CD 8T cells, and many yc-cytokines have been reported to be positively associated with GvHD pathogenesis. The prophylactic use of two IL-2 receptor antagonist antibodies showed a beneficial effect on GvHD in patients with malignant Blood disease after donor-peripheral Blood stem cell transplantation (Fang et al, 2012 Biol Blood Marrow transfer.18: 754-62). Also, the serum levels of IL-15 showed a sharp increase in GvHD patients within the first month after transplantation (Chik et al, 2003, J Pediatr Hematol Oncol.25:960-4), and donor-derived IL-15 was shown to be critical for acute GvHD in the murine GvHD model (Blaser et al, 2005 Blood 105: 894-. Finally, IL-21 expression was observed in skin and colon samples of GvHD patients, but no IL-21 expression was observed in control samples without GvHD, and serum IL-21 levels were elevated in a GvHD murine model, and the use of anti-human IL-21 antibodies reduced weight loss and mortality associated with GvHD after administration (Hippen et al, 2012 Blood 119:619-28, Bucher et al, 2009 Blood 114: 5375-84).

Several embodiments relate to the use of therapeutic antagonist peptides that selectively inhibit IL-15 activity, alone or in combination with other yc-cytokine family members, as therapeutic agents against hair loss and/or hair loss-related disorders. In some embodiments, the antagonist peptide of a customized derivative that selectively inhibits IL-2, IL-15, IL-9, a combination of IL-2 and IL-15, a combination of IL-2 and IL-9, and/or a combination of IL-15 and IL-9 activity is used as a therapeutic agent for treating alopecia and/or alopecia-related disorders. In some embodiments, selectively inhibiting the effect of a combination of IL-2 and IL-15, a combination of IL-2 and IL-9, and/or a tailored derivative of a combination of IL-15 and IL-9 antagonizing the peptide can be additive or synergistic. Several embodiments relate to the use of SEQ ID NO:2 for treating hair loss and/or hair loss related disorders. Several embodiments relate to the use of BNZ-gamma for treating hair loss and/or hair loss related disorders. Several embodiments relate to the use of SEQ ID NO:1 for treating hair loss and/or hair loss related disorders.

Several embodiments relate to the use of therapeutic compounds, alone or in combination, as therapeutic agents for hair loss and/or hair loss related disorders. In some embodiments, the therapeutic compound is SEQ ID NO 2. In some embodiments, the therapeutic compound is BNZ- γ. In some embodiments, the therapeutic compound is SEQ ID NO 1. In some embodiments, the therapeutic compound is an anti-CD 8 antibody. In some embodiments, the therapeutic compound is an anti-IL-2 antibody. In some embodiments, the therapeutic compound is an anti-IL-15 antibody. In some embodiments, the therapeutic compound is an anti-NKG 2A antibody.

Additive effects are observed when the effect of the combination is equal to the sum of the effects of the individual compounds in the combination (e.g., the effect of the combination of two or more therapeutic compounds is equal to the sum of the effects of each therapeutic compound alone). A synergistic effect is observed when the effect of the combination is greater than the sum of the effects of the individual compounds in the combination (e.g., the effect of the combination of two or more therapeutic compounds is greater than the sum of the effects of each therapeutic compound alone). The synergistic effect is greater than the additive effect. Additive effects, synergistic effects, or both can occur in human patients, non-patient human volunteers, in vivo models, ex vivo models, in vitro models, and the like.

In some embodiments, two or more therapeutic compounds disclosed herein can be used in combination. In some embodiments, two or more therapeutic compounds disclosed herein produce an additive effect when used in combination. In some embodiments, two or more therapeutic compounds disclosed herein produce a synergistic effect when used in combination. The synergistic effect may range from about >1 fold to about 100 fold. In some embodiments, the synergistic effect is from about 2-fold to about 20-fold. In some embodiments, the synergistic effect is about 20-fold to about 100-fold. In some embodiments, the synergistic effect is from greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times, or within a range bounded by any two of the aforementioned values.

Another embodiment relates to the development of compounds (non-peptide, non-protein) having a spatial structure resembling the 19-mer amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1) and which can be incorporated into the capsule of the yc-subunit to structurally block the proximity of the yc-cytokine to the yc-subunit for binding. Some embodiments relate to the use of structurally similar compounds as inhibitors of yc-cytokine activity. Such molecular modelling strategies to further improve the development of synthetic compounds structurally similar to existing biological peptides/proteins are described in Orzaez et al, 2009chem.med.chem.4: 146160. Another embodiment relates to the administration of a compound (non-peptide, non-protein) having a 3D structure analogous to the 19-mer amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1) to inhibit, ameliorate, reduce the severity of, treat, delay the onset of, or prevent one or more alopecia and/or alopecia-related disorders.

Several embodiments relate to the administration of a peptide of amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-N-T-S (SEQ ID NO:1) for inhibiting, ameliorating, reducing the severity of one or more alopecia and/or alopecia-related disorders, treating one or more alopecia and/or alopecia-related disorders, delaying the onset of one or more alopecia and/or alopecia-related disorders, or preventing one or more alopecia and/or alopecia-related disorders. Another embodiment relates to the administration of a peptide derived from the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1) for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more hair loss and/or hair loss-related disorders, wherein the amino acid sequence of the derived peptide has the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I- The peptides of N-T-S (SEQ ID NO:1) have similar physicochemical properties but different biological activities. Another embodiment relates to administering to a patient a peptide having the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1) conjugated to the N-and C-terminal or side chain residues of existing bioproteins/peptides for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more hair loss and/or hair loss related disorders.

Several embodiments relate to the administration of a peptide of amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-N-T-S (SEQ ID NO:1) for inhibiting, ameliorating, reducing the severity of one or more alopecia and/or alopecia-related disorders, treating one or more alopecia and/or alopecia-related disorders, delaying the onset of one or more alopecia and/or alopecia-related disorders, or preventing one or more alopecia and/or alopecia-related disorders. Another embodiment relates to the administration of a peptide derivative of the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1) for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more hair loss and/or hair loss-related disorders, wherein the amino acid sequence of the derived peptide has the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I- The peptides of N-T-S (SEQ ID NO:1) have similar physicochemical properties but different biological activities. Another embodiment relates to administering to a patient a peptide having the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1) conjugated to the N-and C-terminal or side chain residues of existing bioproteins/peptides for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more hair loss and/or hair loss related disorders.

Several embodiments relate to the administration of polyclonal and monoclonal antibodies raised against a peptide comprising the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1) as an immunogen to a patient for the purpose of inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more hair loss and/or hair loss related disorders. Another embodiment relates to polyclonal and monoclonal antibodies generated against a derivative peptide of amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1) having amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-I as an immunogen for administration to a patient for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more alopecia and/or alopecia-related disorders, wherein the amino acid sequence of the derivative peptide has amino acid sequence I-K-E-F-L-Q-R-F-I-H-I- The peptides of V-Q-S-I-I-N-T-S (SEQ ID NO:1) have similar physicochemical properties but different biological activities.

Several embodiments relate to the administration of polyclonal and monoclonal antibodies raised against IL-2 as immunogens to a patient for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more hair loss and/or hair loss-related disorders. Another embodiment relates to the administration of polyclonal and monoclonal antibodies raised against IL-15 as immunogens to a patient for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more hair loss and/or hair loss-related disorders. Another embodiment relates to the administration of polyclonal and monoclonal antibodies raised against the transmembrane glycoprotein T-cell co-receptor CD8 to a patient as immunogens for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more hair loss and/or hair loss-related disorders. Another embodiment relates to the administration of polyclonal and monoclonal antibodies raised against members of the NKG2 family of C-type lectin receptors (e.g., NKG2D, NKG2A) as immunogens to a patient for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more hair loss and/or hair loss-related disorders.

Administration of therapeutic compounds

This embodiment also encompasses the use of one or more therapeutic compounds selected from the group consisting of a yc-cytokine antagonist peptide, a yc-cytokine antagonist peptide derivative, an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, or a combination thereof, in the manufacture of a medicament for inhibiting, ameliorating, reducing the severity of, delaying the onset of, or preventing one or more hair loss and/or hair loss-related disorders. This embodiment also encompasses pharmaceutical compositions comprising one or more therapeutic compounds in combination with a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier and a non-toxic therapeutically effective amount of the therapeutic compound, or other compositions of this embodiment.

The present embodiments provide methods of using pharmaceutical compositions comprising an effective amount of a therapeutic compound in a suitable diluent or carrier. The therapeutic compounds of the present embodiments can be formulated according to known methods for preparing pharmaceutically useful compositions. The therapeutic compound may be combined in a mixture as the sole active material or with other known active materials, together with pharmaceutically suitable diluents (e.g., phosphate, acetate, Tris HCl), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifying compounds, solubilizing agents, adjuvants and/or carriers such as bovine serum albumin.

In some embodiments, one or more compositions and kits comprising one or more therapeutic compounds disclosed herein are contemplated. In some embodiments, one or more compositions and kits are used for the prevention and/or treatment of one or more diseases. In some embodiments, one or more compositions and kits are used to inhibit, ameliorate, reduce the severity of, treat, delay the onset of, or prevent one or more hair loss and/or hair loss-related disorders.

In some embodiments, one or more compositions and kits comprising one or more therapeutic compounds are administered to a subject in need thereof via any of the routes of administration provided herein. In some embodiments, one or more compositions and kits comprise a therapeutically effective amount of one or more therapeutic compounds to modulate the signaling of one or more yc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In some embodiments, one or more compositions and kits comprise a therapeutically effective amount of one or more therapeutic compounds to prevent and/or treat one or more diseases. In some embodiments, one or more compositions and kits comprising one or more therapeutic compounds further comprise one or more pharmaceutically acceptable carriers, diluents, excipients, or combinations thereof.

In some embodiments, the one or more therapeutic compounds in the one or more compositions and kits are formulated to be suitable for administration to a subject for the prevention and/or treatment of one or more diseases. In some embodiments, the one or more therapeutic compounds in the one or more compositions and kits are formulated to be suitable for administration to a subject to prevent and/or treat hair loss and/or a hair loss-related disorder.

In some embodiments, one or more therapeutic compounds selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, anti-CD 8 antibodies, anti-IL-2 antibodies, anti-IL-15 antibodies, and anti-NKG 2A antibodies in one or more compositions and kits are formulated as suitable for administration to a subject for the prevention and/or treatment of one or more diseases. In some embodiments, one or more of the composite peptides selected from SEQ ID No. 1, SEQ ID No. 2, anti-CD 8 antibodies, anti-IL-2 antibodies, anti-IL-15 antibodies, and anti-NKG 2A antibodies in one or more compositions and kits are formulated suitable for administration to a subject to inhibit, ameliorate, or reduce the severity of one or more hair loss and/or hair loss associated disorders, treat one or more hair loss and/or hair loss associated disorders, delay the onset of one or more hair loss and/or hair loss associated disorders, or prevent one or more hair loss and/or hair loss associated disorders.

In some embodiments, one or more derivatives of one or more complex peptides selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, and anti-CD 8 antibodies, anti-IL-2 antibodies, anti-IL-15 antibodies, and anti-NKG 2A antibodies in one or more compositions and kits are formulated suitable for administration to a subject for the prevention and/or treatment of one or more diseases. In some embodiments, one or more derivatives of one or more complex peptides selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, anti-CD 8 antibodies, anti-IL-2 antibodies, anti-IL-15 antibodies, and anti-NKG 2A antibodies in one or more compositions and kits are formulated suitable for administration to a subject to inhibit, ameliorate, or reduce the severity of one or more hair loss and/or hair loss associated disorders, treat one or more hair loss and/or hair loss associated disorders, delay the onset of one or more hair loss and/or hair loss associated disorders, or prevent one or more hair loss and/or hair loss associated disorders.

The terms "disease," "disorder," and "biological condition" are used interchangeably in reference to "inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more diseases" provided in accordance with the present embodiments.

In some embodiments, the one or more derivatives of one or more complex peptides comprise an amino acid sequence sharing from about 50% to about 99% identity with the one or more complex peptides. In some embodiments, the one or more derivatives of one or more complex peptides comprise an amino acid sequence that shares 50%, 50-60%, 60-70%, 70-80%, 90%, 95%, 97%, 98%, 99%, or 99.8% identity, or is within a range defined by any two of the above values, with the one or more complex peptides.

In some embodiments, the one or more alopecia-related disorders are selected from: alopecia areata, alopecia totalis, alopecia subtotalis, alopecia universalis, diffuse alopecia, alopecia areata, lichen planus, lichen sclerosus, lichen atrophia sclerosus, atopic dermatitis, psoriasis vulgaris, psoriasis capitis, trichomonad, psoriasis vulgaris, psoriasis retrotropica, psoriatic arthritis, eczema, pemphigus vulgaris, pemphigus foliaceus, pemphigus proliferatum, pemphigus erythematoides, mucosal pemphigoid, scar mucosal pemphigoid, bullous pemphigoid, myasthenia gravis, thyroid disorders, hashimoto's thyroiditis, hypothyroidism, endemic goiter, addison's disease, scleroderma, urticaria, prurigo, rosacea vitiligo, and graft-versus-host disease (GvHD).

Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences,16th ed.1980Mack Publishing CO and a summary of antibody drug delivery (Awwad et al, 2018 pharmaceuticals 10: 83). In addition, such compositions may contain therapeutic compounds complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, and the like, or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroids (spheroblasts). Such compositions will affect the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of the therapeutic compound. The therapeutic compound may be conjugated to an antibody, receptor, ligand directed against a cell-specific antigen, or coupled to a ligand for a tissue-specific receptor.

The method of administering the therapeutic compound of the present embodiment may be appropriately selected depending on factors such as the type of disease, the condition of the subject, and/or the site to be targeted. The therapeutic compound may be administered topically, orally, parenterally, rectally, or by inhalation. Topical application of the therapeutic compound can be achieved by formulation into a lotion, liniment (balm), solution, ointment, cream, paste, gel, or other suitable topical delivery system as appropriate (Gupta et al, 2016Indo Amer J Pharm Res 6: 6353-69). Topical formulation components may include emollients and/or stiffening agents, such as cetyl alcohol, cetyl ester wax, carnauba wax, lanolin alcohols, paraffin, petrolatum, polyethylene glycol, stearic acid, stearyl alcohol, white wax or yellow wax; emulsifiers and/or solubilizers, such as polysorbate 20, polysorbate 80, polysorbate 60, poloxamers, sorbitan monostearate, sorbitan monooleate, sodium lauryl sulfate, propylene glycol monostearate; humectants, such as glycerol, propylene glycol, polyethylene glycol; thickeners/gelling agents, such as carbomers, methylcellulose, sodium carboxymethylcellulose, carrageenan, colloidal silicon dioxide, guar gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, gelatin, polyethylene oxide, alginic acid, sodium alginate, fumed silica; preservatives, such as benzoic acid, propyl paraben, methyl paraben, imidurea, sorbic acid, potassium sorbate, benzalkonium chloride, phenylmercuric acetate, chlorobutanol, phenoxyethanol; penetration enhancers, such as propylene glycol, ethanol, isopropanol, oleic acid, polyethylene glycol; antioxidants, such as butylated hydroxyanisole, butylated hydroxytoluene; buffers such as citric acid, phosphoric acid, sodium hydroxide, sodium dihydrogen phosphate; and vehicle agents such as purified water, propylene glycol, hexylene glycol, oleyl alcohol, propylene carbonate, and mineral oil (Chang et al, 2013AAPS J15: 41-52). Oral formulation components may include fatty acids and derivatives, such as lauric acid, caprylic acid, oleic acid; bile salts such as sodium cholate, sodium deoxycholate, sodium taurodeoxycholate, sodium glycocholate; chelating agents, such as citric acid, sodium salicylate; polymers containing alkyl glycosides, cationic polymers, anionic polymers, and nanoparticles; and surfactants such as sodium lauryl sulfate, sodium dodecyl maltoside laurate, poloxamers, sodium myristate, sodium lauryl sulfate, quillayasaponin, and sucrose palmitate (Liu et al, 2018Expert Opin Drug Del 15:223-33, agiurre et al, 2016Adv Drug release Rev 106: 223-41). The term "parenteral" includes subcutaneous injections, intravenous, intramuscular, intraperitoneal, intracisternal injection or infusion techniques. These compositions generally include an effective amount of a therapeutic compound, either alone or in combination with an effective amount of any other active material. Several non-limiting routes of administration are possible, including parenteral, subcutaneous, intra-articular, intrabronchial, intra-abdominal, intracapsular, intracartilaginous, intracavity, intracavitary, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.

One or more therapeutic compounds disclosed herein can be administered at any dose, via any route of administration, and at any frequency of administration, as determined by one of ordinary skill in the art based on various parameters. Non-limiting examples include the condition being treated, the severity of the condition, patient compliance, efficacy of treatment, side effects, and the like.

The amount of the therapeutic compound contained in the pharmaceutical composition of the present embodiment, the dosage form of the pharmaceutical composition, the frequency of administration, and the like may be appropriately selected depending on factors such as the type of disease, the condition of the subject, and/or the site to be targeted. Such dosages and desired drug concentrations contained in the compositions may vary depending on a number of parameters, including the intended use, the weight and age of the patient, and the route of administration. The preliminary studies will be performed first using animal studies and scaled to human administration according to art accepted practices.

In one embodiment, a host cell that has been genetically modified with a polynucleotide encoding at least one therapeutic compound is administered to a subject for inhibiting, ameliorating, reducing the severity of, treating, delaying the onset of, or preventing one or more alopecia and/or alopecia-related disorders. The polynucleotide is expressed by the host cell, thereby producing the therapeutic compound in the subject. Preferably, the host cell is allogeneic or autologous to the subject.

In another aspect, one or more therapeutic compounds selected from the group consisting of a yc-cytokine antagonist peptide, a yc-cytokine antagonist peptide derivative, an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, or a combination thereof can be used in combination with other therapies (e.g., therapies that inhibit the proliferation and growth of cancer cells). The phrase "combination therapy" includes the administration of one or more therapeutic compounds selected from the group consisting of yc-cytokine antagonist peptides, yc-cytokine antagonist peptide derivatives, anti-CD 8 antibodies, anti-IL-2 antibodies, anti-IL-15 antibodies, anti-NKG 2A antibodies, or combinations thereof, and one or more additional therapeutic agents as part of a particular therapeutic regimen intended to provide a beneficial effect from the combined action of these therapeutic agents. The combined administration of these therapeutic agents is typically carried out over a defined period of time (typically minutes, hours, days or weeks, depending on the combination selected).

Combination therapy is intended to encompass the administration of these therapeutic agents in a sequential manner, i.e., wherein each therapeutic agent is administered at a different time, as well as the administration of these therapeutic agents or at least two therapeutic agents in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple single capsules of each therapeutic agent. Sequential or substantially simultaneous administration of each therapeutic agent can be achieved by appropriate routes, including but not limited to oral routes, intravenous routes, intramuscular routes, and direct absorption through mucosal tissue. The therapeutic agents may be administered by the same route or by different routes. The order of administration of the therapeutic agents is not critical.

Combination therapy may also include the administration of a further combination of a therapeutic agent as described above with other bioactive ingredients (such as, but not limited to, second and different therapeutic agents) and non-drug therapy (such as, but not limited to, surgery or radiation therapy). Where combination therapy also includes radiation therapy, the radiation therapy can be performed at any suitable time so long as the beneficial effects from the combined action of the therapeutic agent and the radiation therapy are achieved. For example, where appropriate, when radiation therapy is temporarily removed from administration of the therapeutic agent, beneficial effects may still be achieved, perhaps over days or even weeks.

In certain embodiments, one or more therapeutic compounds selected from the group consisting of yc-cytokine antagonist peptides, derivatives of yc-cytokine antagonist peptides, anti-CD 8 antibodies, anti-IL-2 antibodies, anti-IL-15 antibodies, anti-NKG 2A antibodies, or combinations thereof, can be administered in combination with at least one antiproliferative agent selected from the group consisting of chemotherapeutic, antimetabolite, and anti-tumorigenic agents, antimitotic, and antiviral agents, and antineoplastic, immunotherapeutic, and radiotherapeutic agents.

In certain embodiments, one or more therapeutic compounds selected from the group consisting of yc-cytokine antagonist peptides, yc-cytokine antagonist peptide derivatives, anti-CD 8 antibodies, anti-IL-2 antibodies, anti-IL-15 antibodies, anti-NKG 2A antibodies, or a combination thereof may be administered in combination with at least one anti-inflammatory agent selected from the group consisting of steroids, corticosteroids, and non-steroidal anti-inflammatory drugs.

Kits for performing any of the above methods are also provided. The kit may comprise one or more therapeutic compounds according to this embodiment selected from the group consisting of a yc-cytokine antagonist peptide, a yc-cytokine antagonist peptide derivative, an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, or a combination thereof. In some embodiments, the kit may comprise instructions. The instructions may be in written or pictorial form, or may be on a recording medium, including audio tape, audio CD, video tape, DVD, CD-ROM, or the like. The kit may comprise a package.

Further embodiments

In some embodiments of the methods, the composite peptide comprises the amino acid sequence D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO:2), wherein X represents any amino acid. In some embodiments of the methods, the composite peptide derivative shares at least about 50% identity with the peptide of SEQ ID No. 2. In some embodiments of the methods, the composite peptide derivative shares at least about 90% identity with the peptide of SEQ ID No. 2. In some embodiments of the methods, the composite peptide derivative shares at least about 95% identity with the peptide of SEQ ID No. 2. In some embodiments of the methods, the composite peptide and the composite peptide derivative have similar physicochemical properties, but different biological activities.

In some embodiments of the methods, the composite peptide comprises the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1) (BNZ- γ). In some embodiments of the methods, the composite peptide derivative shares at least about 50% identity with the peptide of SEQ ID No. 1. In some embodiments of the methods, the composite peptide derivative shares at least about 90% identity with the peptide of SEQ ID No. 1. In some embodiments of the methods, the composite peptide derivative shares at least about 95% identity with the peptide of SEQ ID No. 1. In some embodiments of the methods, the composite peptide and the composite peptide derivative have similar physicochemical properties, but different biological activities.

In some embodiments of the methods, the composite peptide comprises the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO: 1). In some embodiments of the methods, the composite peptide derivative shares at least about 50% identity with the peptide of SEQ ID No. 1. In some embodiments of the methods, the composite peptide derivative shares at least about 90% identity with the peptide of SEQ ID No. 1. In some embodiments of the methods, the composite peptide derivative shares at least about 95% identity with the peptide of SEQ ID No. 1. In some embodiments of the methods, the composite peptide and the composite peptide derivative have similar physicochemical properties, but different biological activities.

In some embodiments of the method, the composite peptide or composite peptide derivative inhibits the activity of one or more yc-cytokines. In some embodiments of the methods, the one or more yc-cytokines are selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In some embodiments of the methods, the composite peptide or composite peptide derivative inhibits the activity of IL-2, IL-15, and IL-9. In some embodiments of the methods, the composite peptide or composite peptide derivative inhibits the activity of IL-2 and IL-15. In some embodiments of the methods, the composite peptide or composite peptide derivative inhibits the activity of IL-15 and IL-9. In some embodiments of the methods, the composite peptide or composite peptide derivative inhibits the activity of IL-15 and IL-21.

In some embodiments, the composite peptide or composite peptide derivative comprises a signal peptide. In some embodiments, the composite peptide or composite peptide derivative is further conjugated to one or more additional moieties on the N-terminal, C-terminal, or side chain residue of the composite peptide or composite peptide derivative. In some embodiments of the composite peptide or composite peptide derivative, the one or more additional moieties are selected from the group consisting of Bovine Serum Albumin (BSA), albumin, Keyhole Limpet Hemocyanin (KLH), the Fc region of an IgG, a biological protein that functions as a scaffold, an antibody directed against a cell-specific antigen, a receptor, a ligand, a metal ion, and polyethylene glycol (PEG).

In some embodiments, the composite peptide or composite peptide derivative comprises at least two α -alkenyl substituted amino acids, and wherein the at least two α -alkenyl substituted amino acids are linked via at least one intrapeptide hydrocarbon linker element. In some embodiments of the composite peptide, the at least two α -alkenyl substituted amino acids are linked by a ring closing metathesis reaction to form at least one intrapeptide hydrocarbon linker element, wherein the ring closing metathesis reaction is catalyzed by a grubbon catalyst.

In some embodiments, the amino acids in the composite peptide are selected from the group consisting of natural amino acids, unnatural amino acids, (D) stereochemically configured amino acids, (L) stereochemically configured amino acids, (R) stereochemically configured amino acids, and (S) stereochemically configured amino acids, and wherein the at least two alpha-alkenyl substituted amino acids are selected from the group consisting of S-pentenylalanine (CAS: 288617-73-2; S5Ala) and R-octenylalanine (CAS: 945212-26-0; R8 Ala).

In some embodiments of the composite peptide, the at least two α -alkenyl substituted amino acids linked by at least one intra-peptide hydrocarbon are separated by n-2 amino acids, where n represents the number of amino acids encompassed within a peptide bond.

In some embodiments of the composite peptide, when the at least two α -alkenyl substituted amino acids linked by the at least one intrapeptide hydrocarbon are separated by three amino acids, the at least one intrapeptide hydrocarbon linker element spans a single α helical turn of the composite peptide.

In some embodiments of the composite peptide, when the composite peptide comprises one or more non-contiguous single alpha helical turns, the amino acid positions associated with the single alpha helical turns of the composite peptide correspond to amino acid positions i and i +4 of the composite peptide, wherein i is the first amino acid position of the single alpha helical turn and i +4 is the last amino acid position of the single alpha helical turn, and wherein amino acid positions i and i +4 comprise alpha-alkenyl substituted amino acids. In some embodiments of the composite peptide, when the α -alkenyl substituted amino acid at position i is S5Ala, the α -alkenyl substituted amino acid at position i +4 is also S5Ala, and the hydrocarbon linker element formed by the ring closing metathesis reaction is represented by formula 1.

In some embodiments of the composite peptide, when the at least two α -alkenyl substituted amino acids linked by the at least one intrapeptide hydrocarbon are separated by 6 residues, the at least one intrapeptide hydrocarbon linker element spans the double α helical turn of the composite peptide.

In some embodiments of the composite peptide, when the composite peptide comprises one or more non-contiguous double alpha helical turns, the amino acid positions associated with the double alpha helical turns of the composite peptide correspond to amino acid positions i and i +7 of the composite peptide, wherein i is the first amino acid position of the double alpha helical turn and i +7 is the last amino acid position of the double alpha helical turn, and wherein amino acid positions i and i +7 comprise alpha-alkenyl substituted amino acids. In some embodiments of the composite peptide, when the α -alkenyl substituted amino acid at position i is R8Ala, the α -alkenyl substituted amino acid at position i +7 is S5Ala, and the hydrocarbon linker element formed by ring closing metathesis is represented by formula 2.

In some embodiments, the composite peptide comprises an amino acid sequence of at least two D-helical regions of a γ -c-box of an Interleukin (IL) protein, wherein the composite peptide comprises the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-N-T-S (SEQ ID NO:1), and wherein the composite peptide comprises at least two α -alkenyl substituted amino acids, and wherein the at least two α -alkenyl substituted amino acids are linked via at least one intra-peptide hydrocarbon linker element.

In some embodiments, the composite peptide comprises an amino acid sequence of at least two D-helical regions of a γ -c-box of an Interleukin (IL) protein, wherein the composite peptide comprises the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-N-T-S (SEQ ID NO:1), and wherein the composite peptide comprises at least two α -alkenyl substituted amino acids, and wherein the at least two α -alkenyl substituted amino acids are linked via at least one intra-peptide hydrocarbon linker element.

In some embodiments of the composite peptide, one or more carbon-carbon double bonds present in the hydrocarbon linker within the peptide are used in one or more organic chemical reactions to add one or more additional chemical functional groups. In some embodiments of the composite peptide, the one or more organic chemical reactions comprise an alkene reaction. In some embodiments of the composite peptide, the alkene reaction is selected from hydroboration, hydromercurization, hydration, chlorination, bromination, addition of HF, HBr, HCl or HI, dihydroxylation, epoxidation, hydrogenation, and cyclopropanation. In some embodiments of the composite peptide, one or more additional chemical functional groups may be added after the olefin reaction, wherein the one or more additional chemical functional groups comprise covalent addition of one or more chemical group substituents, wherein the covalent addition of one or more chemical group substituents comprises a nucleophilic reaction with an epoxide and a hydroxyl group. In some embodiments of the composite peptide, the one or more additional chemical functional groups are selected from the group consisting of biotin, a radioisotope, a therapeutic agent, rapamycin, vinblastine, paclitaxel, a non-protein fluorescent chemical group, FITC, hydrazide, rhodamine, maleimide, a protein fluorescent group, GFP, YFP and mCherry.

In some embodiments, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the peptide conjugate or derivative thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof, wherein the peptide conjugate or derivative thereof modulates the activity of two or more yc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, wherein the peptide conjugate comprises the amino acid sequence D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO:2), wherein X represents any amino acid, and wherein the derivative thereof comprises a sequence identical to SEQ ID NO:2, or a peptide sequence sharing at least 90% identity with the amino acid sequence of seq id No. 2.

In some embodiments, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the peptide conjugate or derivative thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof, wherein the peptide conjugate or derivative thereof modulates the activity of two or more yc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, wherein the peptide conjugate comprises the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1), and wherein the derivative thereof comprises a sequence identical to SEQ ID NO:1, or a peptide sequence sharing at least 90% identity with the amino acid sequence of 1.

In some embodiments, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the peptide conjugate or derivative thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof, wherein the peptide conjugate or derivative thereof modulates the activity of two or more yc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, wherein the peptide conjugate comprises the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1), and wherein the derivative of the peptide conjugate comprises a peptide sequence identical to SEQ ID NO:1, or a peptide sequence sharing at least 90% identity with the amino acid sequence of 1.

In some embodiments of the pharmaceutical composition, the peptide conjugate or derivative thereof inhibits the activity of two or more yc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In some embodiments of the pharmaceutical composition, the peptide conjugate or derivative thereof further comprises an additional conjugate at the N-terminus, C-terminus, or a side chain residue thereof.

In some embodiments of the pharmaceutical composition, the peptide conjugate or derivative thereof further comprises a signal peptide. In some embodiments, the pharmaceutical composition further comprises a protein that stabilizes the structure and increases the biological activity of the peptide conjugate or a derivative thereof, wherein the protein is selected from the group consisting of Bovine Serum Albumin (BSA), albumin, an Fc region of immunoglobulin g (igg), a biological protein that functions as a scaffold, polyethylene glycol (PEG), and derivatives thereof. In some embodiments of the pharmaceutical composition, the derivative thereof comprises a peptide sequence sharing at least 95% identity with the amino acid sequence of SEQ ID No. 2. In some embodiments of the pharmaceutical composition, the derivative thereof comprises a peptide sequence sharing at least 95% identity with the amino acid sequence of SEQ ID No. 1.

In some embodiments, methods of treating alopecia-related disorders are provided. In some embodiments, the method comprises administering to a subject in need thereof a pharmaceutical composition provided herein, wherein the alopecia-associated disorder is selected from: alopecia areata, alopecia totalis, alopecia subtotalis, alopecia universalis, diffuse alopecia, alopecia areata, lichen planus, lichen sclerosus, lichen atrophia sclerosus, atopic dermatitis, psoriasis vulgaris, psoriasis capitis, trichomonad, psoriasis vulgaris, psoriasis retrotropica, psoriatic arthritis, eczema, pemphigus vulgaris, pemphigus foliaceus, pemphigus proliferatum, pemphigus erythematoides, mucosal pemphigoid, scar mucosal pemphigoid, bullous pemphigoid, myasthenia gravis, thyroid disorders, hashimoto's thyroiditis, hypothyroidism, endemic goiter, addison's disease, scleroderma, urticaria, prurigo, rosacea vitiligo, and graft-versus-host disease (GvHD).

In some embodiments, a kit for treating a hair loss-related disorder in a patient is provided.

In some embodiments, the kit comprises a pharmaceutical composition, wherein the pharmaceutical composition comprises a therapeutically effective amount of the peptide conjugate or derivative thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof, wherein the peptide conjugate or derivative thereof modulates the activity of two or more yc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, wherein the peptide conjugate comprises the amino acid sequence D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO:2), wherein X represents any amino acid, and wherein the derivative thereof comprises a sequence identical to SEQ ID NO:2, or a peptide sequence sharing at least 90% identity with the amino acid sequence of seq id No. 2.

In some embodiments, the kit comprises a pharmaceutical composition, wherein the pharmaceutical composition comprises a therapeutically effective amount of the peptide conjugate or derivative thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof, wherein the peptide conjugate or derivative thereof modulates the activity of two or more yc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, wherein the peptide conjugate comprises the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1), and wherein the derivative thereof comprises a sequence identical to SEQ ID NO:1, or a peptide sequence sharing at least 90% identity with the amino acid sequence of 1.

In some embodiments, the kit comprises a pharmaceutical composition, wherein the pharmaceutical composition comprises a therapeutically effective amount of the peptide conjugate or derivative thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof, wherein the peptide conjugate or derivative thereof modulates the activity of two or more yc-cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, wherein the peptide conjugate comprises the amino acid sequence I-K-E-F-L-Q-R-F-I-H-I-V-Q-S-I-I-N-T-S (SEQ ID NO:1), and wherein the derivative thereof comprises a sequence identical to SEQ ID NO:1, or a peptide sequence sharing at least 90% identity with the amino acid sequence of 1.

In some embodiments of the kit, the condition is one or more of: alopecia areata, alopecia totalis, alopecia subtotalis, alopecia universalis, diffuse alopecia, alopecia areata, lichen planus, lichen sclerosus, lichen atrophia sclerosus, atopic dermatitis, psoriasis vulgaris, psoriasis capitis, trichomonad, psoriasis vulgaris, psoriasis retrotropica, psoriatic arthritis, eczema, pemphigus vulgaris, pemphigus foliaceus, pemphigus proliferatum, pemphigus erythematoides, mucosal pemphigoid, scar mucosal pemphigoid, bullous pemphigoid, myasthenia gravis, thyroid disorders, hashimoto's thyroiditis, hypothyroidism, endemic goiter, addison's disease, scleroderma, urticaria, prurigo, rosacea vitiligo, or graft-versus-host disease (GvHD).

Definition of

As used herein, the term "patient" or "subject" refers to the recipient of any embodiment of the composite peptides disclosed herein, and includes all organisms within the kingdom animalia. In some embodiments, any vertebrate animal is included, including but not limited to humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cows, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild, and game birds such as chickens, turkeys, and other gallinaceous birds, ducks, geese, and the like). In a preferred embodiment, the animal belongs to the mammalian family, such as humans, bovines, ovines, porcines, felines, buffalos, canines, caprines, equines, donkeys, deer and primates. The most preferred animal is a human. In some embodiments, the patient is male or female.

As used herein, the term "treatment" or any variant thereof (e.g., treatment), treatment, etc.) refers to any treatment of a patient diagnosed with a biological condition, such as alopecia areata, alopecia totalis, sub-alopecia totalis, alopecia universalis, diffuse alopecia, alopecia areata, lichen planus, lichen sclerosus atrophica, atopy, atopic dermatitis, psoriasis vulgaris, psoriasis scalp psoriasis, guttate psoriasis, parapsoriatic psoriasis, psoriatic arthritis, eczema, pemphigus vulgaris, pemphigus foliaceus, pemphigus proliferatum, pemphigus erythematoides, mucocutaneous pemphigoid, mucocutaneous cicatricial pemphigoid, bullous pemphigoid, myasthenia gravis, thyroid disorders, hashimoto's thyroiditis, hypothyroidism, endemic goiter, adenitis, etc, Localized scleroderma, urticaria, prurigo, rosacea, vitiligo, and graft-versus-host disease (GvHD).

The term treatment as used herein includes: (i) preventing or delaying the presentation of symptoms associated with a target biological condition in an at-risk patient to show symptoms associated with the biological condition; (ii) ameliorating a symptom associated with a target biological condition in a patient diagnosed with the biological condition; (iii) preventing, delaying or ameliorating the presentation of complications, conditions or disease-related symptoms associated with a target biological condition in a patient at risk or a patient diagnosed with a biological condition; (iv) slowing, delaying or halting the progression of the biological condition; and/or (v) preventing, delaying, slowing, halting or ameliorating a cellular event of inflammation; and/or (vi) other clinical measures to prevent, delay, slow, halt, or ameliorate histological abnormalities and/or biological conditions.

The term "symptom" as used herein refers to a common sign or indication that a patient is suffering from a particular condition or disease.

The term "effective amount" as used herein refers to the amount required to elicit a desired biological response. According to this embodiment, an effective amount of a yc-antagonist is the amount required to provide an observable effect of at least one biological factor for treating a biological condition.

"recombinant DNA technology" or "recombinant" refers to the use of techniques and methods for producing a particular polypeptide from a microorganism (e.g., bacteria, yeast), invertebrate (insect), mammalian cell or organism (e.g., transgenic animal or plant) that has been transformed or transfected with cloned or synthetic DNA sequences to enable biosynthesis of a heterologous peptide. Natural glycosylation patterns can only be achieved with mammalian cell expression systems. Prokaryotic expression systems lack the ability to add glycosylation to the synthesized protein. Yeast and insect cells provide unique glycosylation patterns that may differ from the native patterns.

"nucleotide sequence" refers to a polynucleotide in the form of an individual fragment or as a component of a larger DNA construct derived from DNA or RNA isolated at least once in substantially pure form, free of contaminating endogenous material, and in an amount or concentration that enables identification, manipulation and recovery of its component nucleotide sequences by standard Molecular Biology methods, as outlined in Current Protocols in Molecular Biology.

"recombinant expression vector" refers to a plasmid comprising a transcriptional unit comprising the following assembly: (1) genetic elements having a regulatory effect on gene expression, including promoters and enhancers, (2) structural or coding sequences encoding polypeptides according to this embodiment, and (3) appropriate transcription and translation initiation sequences, and, if desired, termination sequences. Structural elements intended for use in yeast and mammalian systems preferably include signal sequences that enable extracellular secretion of the translated polypeptide by a yeast or mammalian host cell.

By "recombinant microbial expression system" is meant a substantially homologous single culture of a suitable host microorganism, e.g., a bacterium such as e.coli (e.coli) or yeast such as saccharomyces cerevisiae (s.cerevisiae), which has stably integrated the recombinant transcriptional unit into the chromosomal DNA or carries the recombinant transcriptional unit as a component of a residual plasmid. Typically, the host cell constituting the recombinant microbial expression system is the progeny of a single ancestral transformed cell. The recombinant microbial expression system will express the heterologous polypeptide after induction of regulatory elements linked to the structural nucleotide sequence to be expressed.

As used herein, the section headings are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, treatises, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. The definitions provided in the present teachings will prevail when the definitions of terms in the incorporated references appear to differ from those provided in the present teachings. It is understood that there is an implied "about" before the temperatures, concentrations, times, etc. discussed in the present teachings such that slight and insubstantial deviations are within the scope of the present teachings herein.

Although the present invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses of the invention and obvious modifications and equivalents thereof. In addition, while several variations of the invention have been shown and described in detail, other modifications, which are within the scope of this invention, will be readily apparent to those of skill in the art based upon this disclosure.

Also included are various combinations or subcombinations of the specific features and aspects of the embodiments that may be made and still fall within the scope of the invention. It should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes or embodiments of the disclosed inventions. Accordingly, it is intended that the scope of the invention herein disclosed should not be limited by the particular disclosed embodiments described above.

It should be understood, however, that the detailed description, while indicating preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art.

Examples

The following examples are presented for illustrative purposes and should not be construed as limiting.

Example 1-method for evaluating inhibitory Activity of yc-antagonistic peptide

The proliferative response to a member of the yc-cytokine family is measured using a mammalian cell assay to determine the ability of any custom-derived peptide prepared according to this embodiment to inhibit the action of a member of the yc-cytokine family.

For each of the 6 yc-cytokines, the indicator cell lines were transfected with the human IL-2R β gene: NK92 (a human NK cell line NK92 (catalog number CRL-2407) available through the American Type Culture Collection, ATCC), CTLL-2 (a murine CD 8T cell line available from ATCC), and PT-18 (a murine mast cell line) and its subclone PT-18 β, to allow the cells to respond to IL-2 and IL-15(Tagaya et al, 1996, EMBO J.15:4928-39) and to be used for the quantitative determination of the growth promoting activity of yc-cytokines (for method reference, see Current protocols in Immunology from Wilroom and Sons). When measured at a range of concentrations by the colorimetric WST-1 assay, the indicator cells exhibit a semi-linear dose-dependent response (see Clontech PT3946-1 and related user manual for a detailed description of reagents and methods, which is incorporated herein by reference).

Once the appropriate dose of cytokine from the indicator cell line producing 50%% and 95% maximal response was determined, purified or synthetic custom-derived peptides at various concentrations (ranging from 1pM to 10 μ M) were added to each well containing cytokine and indicator cells. The decrease in absorbance at 450nm was used as an index for inhibiting cytokine-stimulated cell proliferation. Typically, cells are stimulated by cytokines such that the absorbance of wells containing the indicator cell line and cytokine is from 2.0 to 3.0, which is reduced to a range of 0.1 to 0.5 by the addition of an inhibitory peptide.

Example 2-Selective inhibition of the growth promoting Activity of certain yc-cytokines by BNZ-gamma

The ability of BNZ-gamma peptides to specifically inhibit the growth promoting activity of selected yc-cytokines was determined using PT-18 beta cells as described above (fig. 3A). IL-3, a non-yc-cytokine that supports growth of PT-18 beta cells, was used as a negative control. Briefly, PT-18 beta cells were incubated with two different dilutions of BNZ-gamma peptide produced by HEK293T cells (1: 20 or 1:60 dilution of the original supernatant of HEK293T cells transfected with a BNZ-gamma expression construct) or in the absence of BNZ-gamma peptide in the presence of IL-3, IL-9, IL-15 or IL-4 (1 nM of various cytokines in culture).

The growth response of the cells was determined 2 days after the BNZ-gamma peptide and cytokine introduction using the WST-1 assay. The growth promoting activity of IL-3, a non-gammac cytokine, was not inhibited by BNZ-gamma. In contrast, the activity of IL-15 and IL-9 was significantly reduced by BNZ-gamma peptide (p <0.01, student T test). Cell proliferation stimulated by IL-4 (another yc-cytokine) was not affected by the addition of BNZ-gamma peptide. The results for IL-3, IL-9, IL-15 and IL-4 are shown in FIG. 3A.

In a similar assay, the murine cell line CTTL2 was used. In this assay, cells were cultured using 0.5nM recombinant IL-2 in RPMI 10% fetal calf serum. To establish a proliferation assay, cells were washed 3 times from cytokines. Cells were seeded at a density of 1x 10(5) cells per well of a 96-well plate, with a final concentration of IL-2 or IL-15 of 50 pM. Different concentrations of BNZ-gamma peptide (0.1, 1 and 10. mu.M) were added to each well. Cells were cultured for 20 hours and at the last 4 hours, plates were added³H-thymidine. Cells were harvested and radioactivity was measured to determine the level of cell proliferation. The data are shown in fig. 3B.

Example 3 measurement of inhibition of yc-cytokines by measuring 3H-thymidine incorporation as a marker of cell proliferation Method of bioactivity

Inhibition of yc-cytokine induced proliferation of indicator cell populations by antagonist-tailored derivative peptides was measured by a 3H-thymidine incorporation assay. Briefly, radiolabeled thymidine (1 micci) was administered to 20-50,000 cells undergoing proliferation in the presence of cytokines. Radioactivity incorporated by the cells was measured by capturing cell-bound radioactivity on a glass fiber filter using a conventional capture instrument (e.g., Filtermate Universal Harvester from Perkin-Elmer), and then measured using a b-counter (e.g., 1450Trilux microplate scintillation counter).

Example 4 measurement of inhibition of γ by determining incorporation of cell tracking dyes as markers of cell proliferation Methods of c-cytokine activity

Incubating the indicator cells in the presence of the selected yc-cytokine or in the presence of the selected yc-cytokine and the selected custom-derived peptide. The cell population is then labeled in vitro with a cell tracking dye, such as CMFDA from Invitrogen, C2925, and the decay in green fluorescence of the cells at each cell division is monitored using a flow cytometer (e.g., Beckton-Dickinson FACScalibur). Typically, in response to yc-cytokine stimulation, 7-10 distinct peaks will appear on the green fluorescence channel corresponding to the number of divisions a cell has undergone. Depending on the extent of inhibition, incubation of cells with the selected yc-cytokine and antagonist custom-derived peptide reduced the number of peaks to only 1-3.

Example 5 inhibition of intracellular signalling by tailoring peptide derivative antagonists

In addition to stimulating cell proliferation, the binding of yc-cytokines to their receptors also causes different intracellular events (Rochman et al, 2009Nat. Rev. Immunol.9:480-90, Pesu et al, 2005 Immunol.Rev.203: 127-142). Shortly after the cytokine binds to its receptor, the tyrosine kinase known as Jak3 (Janus kinase 3) is recruited to the receptor at the plasma membrane. The kinase phosphorylates tyrosine residues of a variety of proteins, including the yc-subunit, STAT5 (a signal transducer and activator of transcription 5), and subunits of PI3 (phosphatidylinositol 3) kinase. Of these, phosphorylation of STAT5 has been shown in many studies to be associated with yc-cytokine initiated cell proliferation. (reviewed in Hennighausen and Robinson,2008 Genes Dev.22: 711-21). Based on these published data, it was examined whether the BNZ-gamma peptide inhibits tyrosine phosphorylation of the STAT5 molecule in PT-18 β cells stimulated by IL-15 (results shown in fig. 4).

PT-18 beta cells are stimulated by IL-15 in the presence or absence of BNZ-gamma peptide. Cytoplasmic proteins were extracted from cells according to conventional methods as described in Tagaya et al, 1996EMBO J.15: 4928-39. The extracted cytoplasmic proteins were resolved using standard SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the phosphorylation status was confirmed by anti-phospho-STAT 5 antibody (Cell Signaling Technology, catalog number 9354, Danvers MA) using immunoblotting (see figure 4, upper panel). To confirm that each lane represents a similar total protein load, the membrane was then stripped and re-probed with an anti-STAT 5 antibody (Cell Signaling Technology, catalog number 9358) (see figure 4, lower panel).

These results demonstrate that tyrosine phosphorylation of STAT5 (a marker of signaling) is induced by IL-15 in PT-18 β cells, and that tyrosine phosphorylation of STAT5 is significantly reduced by BNZ- γ peptide.

EXAMPLE 6 rational design of derivatives of gammac-antagonistic peptides

The derived peptides were prepared by substituting the defined amino acids of the core sequence with amino acids having the same physicochemical properties as specified in FIG. 2, based on the core sequence D/E-F-L-E/Q/N-S/R-X-I/K-X-L/I-X-Q (SEQ ID NO:2), wherein X represents any amino acid.

Alternatively, custom peptides or peptides derived therefrom may be prepared based on sequence alignment of the D-helix regions of different yc-cytokine family members.

Example 7-method for identifying the inhibitory specificity of antagonistic custom-derived peptides

The yc-cytokine inhibitory specificity of the antagonistic custom-derived peptides was determined by measuring the ability of the custom-derived peptides to inhibit the proliferative response of cytokine-responsive cell lines to each yc-cytokine. For example, the mouse cell line CTLL-2 was used to determine whether candidate peptides inhibited the function of IL-2 and IL-15. PT-18 (. beta.) cells were used to determine whether candidate peptides inhibited the function of IL-4 and IL-9. PT-18(7 α) cells were used to determine whether the candidate peptide inhibited the function of IL-7, and PT-18(21 α) cells were used to determine whether the candidate peptide inhibited the function of IL-21. PT-18 (. beta.) represents a subclone of PT-18 cells exogenously expressing human IL-2 R.beta.by gene transfection (see Tagaya et al, 1996), PT-18 (7. alpha.) represents a subclone expressing human IL-7 R.alpha.by gene transfection, and PT-18(21 R.alpha.) cells express human IL-21 R.alpha..

Another alternative is to use other cell lines that respond to a range of cytokines. An example of this cell line among the human NK cell line NK92 is commercially available from ATCC (catalog number CRL-2407). This cell line is an IL-2 dependent cell line which responds to other cytokines including IL-9, IL-7, IL-15, IL-12, IL-18, IL-21(Gong et al, 1994Leukemia8: 652-.

EXAMPLE 8 preparation of gammac-antagonistic peptide

The derivative yc-antagonistic peptide is custom-made by chemical synthesis through manual or automated processes.

Manual synthesis: classical liquid phase synthesis is employed which involves coupling the carboxyl or C-terminus of one amino acid to the amino or N-terminus of another amino acid. Alternatively, Solid Phase Peptide Synthesis (SPPS) is used.

Automated synthesis: many commercial companies offer automated peptide synthesis at a cost. These companies use various commercial peptide synthesizers, including the synthesizer supplied by Applied Biosystems (ABI). The custom-made derivative gammac-antagonistic peptide is synthesized by an automated peptide synthesizer.

Example 9 biological Generation of custom-derived yc-antagonistic peptides Using recombinant techniques

The yc-antagonistic peptide is custom-derived from propeptide biosynthesis, consisting of an appropriate tag peptide, signal peptide or peptide derived from a known human protein that enhances or stabilizes the structure of the BNZ-gamma peptide and improves its biological activity. If desired, an appropriate enzymatic cleavage sequence prior to the N-terminus of the peptide should be designed to remove the tag or any portion of the peptide from the final protein.

The nucleotide sequence encoding the custom-derived peptide with a stop codon at the 3' end was inserted into a commercial vector having a tag portion of thioredoxin derived from e.coli and a specific peptide sequence recognized and digested by an appropriate proteolytic enzyme (e.g., enterokinase) interposed between the tag portion and the nucleotide sequence encoding the custom-derived peptide and the stop codon. An example of a suitable vector is the pThioHis plasmid available from Invitrogen, CA. Other expression vectors may be used.

Example 10 conjugation of tailored peptides and derivatives to Carrier proteins for immunization purposes and Generation of peptides tailored thereto Antibodies

BNZ-gamma or a derivative thereof is used to immunize animals to obtain polyclonal and monoclonal antibodies. Peptides are conjugated to the N-terminus or C-terminus of an appropriate carrier protein (e.g., bovine serum albumin, Keyhole Limpet Hemocyanin (KLH), etc.) by conventional methods using glutaraldehyde or m-maleimidobenzoyl-N-hydroxysuccinimide ester. The conjugated peptide, in combination with a suitable adjuvant, is then used to immunize an animal such as a rabbit, rodent or donkey. The specificity of the resulting antibodies was checked using conventional methods. If the resulting antibody is reactive with an immunogenic peptide, it is then tested for its ability to inhibit yc-cytokine activity alone according to the cell proliferation assay described in examples 1-3. Due to the complex nature of the derived peptides, it is possible to generate a single antibody that recognizes two different cytokines simultaneously, due to the complex nature of these peptides.

Example 11 Large Scale production of custom-derived yc-antagonistic peptides

Recombinant proteins are produced on a large scale by using cell-free systems as described elsewhere. (see Takai et al, 2010 Curr. pharm. Biotechnol.11(3): 272-8). Briefly, cDNAs encoding a yc-antagonistic peptide and a tag are subcloned into an appropriate vector (see Takai et al, 2010 Curr. pharm. Biotechnol.11(3):272-8), transcribed in vitro, and immediately translated in vitro to produce the labeled peptide. The pre-polypeptide was then purified using an immobilized antibody recognizing the tagged epitope, treated by proteolytic enzymes, and the purity of the eluate (which contained predominantly the target custom-derived peptide) was tested using conventional 18% Tricine-SDS-page (invitrogen) and conventional coomassie staining. If the desired peptide purity (> 98%) is not met, the mixture is subjected to conventional HPLC (high performance liquid chromatography) for further purification.

Example 12 use of a humanized NSG mouse model for therapeutic study of immune-mediated alopecia and alopecia-related disorders Use of

The major progress in the in vivo studies of the human immune system was the development of a DNA that could be used in severely immunodeficient mice such as immunocompromised NOD/Scid/Il2rg^-/-(NSG) the functional human immune system is established in mice (Shultz et al, 2012 Nat. Rev. Immunol.12: 786-98). NSG mice lack the functional yc-subunit required for yc-cytokine signaling, lymphoid cells are extremely deficient, and allow very efficient transplantation of the human immune system following intraperitoneal administration of Ficoll gradient purified human peripheral blood mononuclear cells (hupmc). Subsequent expansion of human immune cells leads to a humanized mouse model of systemic graft versus host disease (GvHD) because human T cells target murine tissues including the skin (Sonntag et al, 2015j. autoimmun.62: 55-66). Humanized NSG mice develop progressive hair loss (alopecia) as a symptom of systemic GvHD, which develops into complete hair loss after about 3-4 weeks, and develop complete hair loss in about 45-50 days. Animals die quickly due to GvHD.

To further understand the underlying mechanism of hair loss in the humanized mouse model, expression profiles of three key circulating human cytokines (IL-2, IL-15, and IFN γ) for hair loss were characterized following intraperitoneal administration of 2 million hupMC to 5 3-week-old NSG mice. The increase in IL-15 was the earliest and evident on day 14, whereas IL-2 and IFN γ did not rise until day 35, with all three cytokines increasing to day 49 (results shown in FIG. 5), which is the last time point available due to death of mice in the experimental group. This suggests that IL-15 is a key driver of disease. By day 35, mice showed symptoms of GvH response, including weight loss and moderate to severe hair loss.

Example 13 Effect of anti-human CD8 antibodies on humanized NSG mice with immune-mediated alopecia

Members of the NKG2 family are involved in the cytotoxic processes of NK and CD8+ T cells and are regulated by a variety of cytokines, including the yc-cytokine IL-15 (Borrego et al, 1998J Exp Med 187:813-18, Brumbaugh et al, 1996J Immunol 157:2804-12, Cantoni et al, 1998Eur J Immunol 28:327-38, Mingari et al, 1998 Proc Natl Acad Sci 95: 1172-7). Each NKG2 receptor dimerizes with the lectin protein CD94 to form a heterodimeric receptor complex (Lazetic et al, 1996J Immunol 157:4741-5), except that NKG2D exists as a homodimer (Garrity et al, 2005 Proc Natl Acad Sci102: 7641-6). Previous reports indicate that hair loss in patients with hair loss is mediated by cytotoxic CD8+ T cells expressing the NKG2D receptor (Xing et al, 2014Nat Med 9:1043-9, Gilhar et al, 2016 autoimmun. rev.15: 726-35). To characterize the importance of CD8+ T cells in this disease model, animals were treated with anti-human CD8 antibody depleted of human CD8+ T cells (OKT8) (BD Biosciences). A cohort of 5 mice developed weight loss and patchy to complete hair loss within 4 weeks after 2 million HuPBMCs had been transplanted. Three humanized mice were then selected for treatment with two injections (twice per week) of 50 μ g/mouse anti-CD 8 antibody.

Prior to treatment with anti-human CD8 antibody, human CD8+ T cells were isolated from blood samples collected from representative humanized NSG mice and stained for expression of NKG2D (CD314) receptors and receptors in the NKG2 family (NKG2A and NKG2C) to facilitate measurement by flow cytometry. Cytotoxic CD8+ T cells in the progression of alopecia disease were also characterized as positive for expression of the activated NKG2D receptor (Xing et al, 2014Nat Med 9: 1043-9). Flow cytometry revealed that almost the entire human CD8+ T cell population isolated from the humanized NSG mice was NKG2D + (see figure 6A). Interestingly, although human NKG2C + CD8+ T cells were observed to decrease after huPBMC transplantation, human NKG2A + CD8+ T cells showed a significant increase after huPBMC transplantation, which only expanded with worsening GvHD symptoms and disease progression (see figure 6B).

After treatment with anti-human CD8 antibody, all human CD8+ T cells were significantly and specifically depleted (see fig. 7A), which did not reappear after treatment. All three humanized mice showed weight gain within 4 days after CD8+ T cell depletion (see fig. 7B), and regrowth of body hair was evident two weeks after treatment (see fig. 7C).

Example 14 with ReversalComposition of human NKG2A + CD8+ T cells in humanized NSG mice with epidemic-mediated alopecia Type gammac-signalling

The interaction between yc-subunit and yc-cytokine results in the activation and phosphorylation of Jak 3. Considering that the interaction of the yc-subunit with Jak3 is very specific, since no other receptor molecules recruit Jak3 for signaling, next it was tested whether human NKG2A + CD8+ T cells isolated from humanized NSG mice 4 weeks after 2 million Hupbmc transplants were positive for phosphorylation of Jak3 and downstream phosphorylation of STAT 5. Human NKG2A + and NKG2A-CD8+ T cells were Ficoll purified from blood and spleen of three representative humanized NSG mice. The cells were then stained with a mixture of FITC-anti-CD 4, PE-anti-CD 8, and PE/Cy 7-anti-NKG 2A, and fluorescence-activated cells were sorted (FACSAria II, BD Biosciences) into CD4-CD8+ NKG2A + and CD4-CD8+ NKG 2A-subsets. As a control, non-transplanted NKG2A + and NKG2A-CD8+ T cells remained unstimulated or stimulated by ex vivo addition of IL-15. Cytoplasmic proteins were extracted from cells according to the general method described by Tagaya et al, 1996EMBO J.15: 4928-39. The extracted cytoplasmic proteins were resolved using standard SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the phosphorylation status was confirmed by anti-phosphorylation-Jak 3 antibody (Cell Signaling Technology, cat # 5031, Danvers MA) or anti-phosphorylation-STAT 5 antibody (Cell Signaling Technology, cat # 9354, Danvers MA) using immunoblotting (see fig. 8). Focal adhesion proteins were explored as controls. The results show constitutive yc-signaling in humanized NSG mice 4 weeks after transplantation of 2 million hupmc by human NKG2A + instead of NKG2A-CD8+ T cells.

Example 15 human C-type lectin in CD8+ T cells of humanized NSG mice with immune-mediated alopecia Antibody-mediated depletion of receptor NKG2 family members

To test the pathogenic involvement of human C-type lectin receptor NKG2 family members (NKG2A, B, C, D, E, F and H) in the pathogenesis of systemic GvHD in humanized NSG mice in CD8+ T cells, antibody-mediated depletion of each human NKG2 protein member in CD8+ T cells was performed by injecting 50 μ g/mouse of anti-NKG 2 antibody specific for the NKG2 protein member in the study twice weekly in three representative humanized NSG mice 3-5 weeks after 2 million huPBMC transplants. Successful depletion of specific NKG2 family members in CD8+ T cells was then associated with major systemic GvHD symptoms (such as weight loss, immune-mediated alopecia and circulating levels of cytokines IL-2, IL-15 and IFN γ).

Example 16-antibody-mediated depletion of human NKG2A + CD8+ T cells on human sources with immune-mediated alopecia Effect of chemosynthesis of NSG mice

To further examine whether NKG2A + CD8+ T cells were causally related to systemic GvHD symptoms such as weight loss and hair loss observed after transplantation of huPBMC in NSG mice, a compilation of kinetics of NKG2A + CD8+ T cell expansion and weekly body weights and levels of inflammatory cytokines IFN γ and γ c cytokines IL-2, IL-7 and IL-15 from three representative humanized NSG mice 1 to 6 weeks after 2 million huPBMC transplants was generated. A clear correlation was observed between the increase of NKG2A + cells in the CD8+ T cell compartment and the increase of IL-2, IL-15 and IFN γ (see FIG. 9A).

To test the pathogenic involvement of NKG2A + CD8+ T cells in the pathogenesis of systemic GvHD in humanized NSG mice, antibody-mediated depletion of human NKG2A + CD8+ T cells was performed by twice weekly injection of 50 μ g/mouse of anti-NKG 2A antibody (R & DSystems, catalog No. MAB1059, Clone 131411, Minneapolis, MN) in three representative humanized NSG mice 3 to 5 weeks after 2 million huPBMC transplants. Successful depletion of NKG2A + CD8+ T cells (see figure 9B, 4-6 weeks post huPBMC transplantation) was positively correlated with reduction of major systemic GvHD symptoms, weight loss and immune-mediated hair loss improvement after one week of anti-NKG 2A antibody treatment. It was observed that the reduction of IL-2, IL-15 and IFN γ was directly related to antibody-mediated depletion of human NKG2A + CD8+ T cells (see FIG. 9B).

Example 17 Effect of BNZ-gamma on humanized NSG mice with immune-mediated alopecia

To test the effects of BNZ- γ, five humanized NSG mice were allowed to develop extensive GvHD with generalized hair loss prior to initiation of treatment (4 weeks after approximately 2 million huPBMC transplants). All animals appeared very sick when treated Intravenously (IV) twice weekly for 2 weeks starting with PEGylated BNZ-gamma (day 0, 2 mg/kg). Control PBS-treated animals died within about 1-2 weeks. On day 21, BNZ- γ treated animals gained significant body weight, with skin appearing healthier, and their fur visibly regrowth. The effect of BNZ- γ lasted for about 2 weeks after completing the two week treatment, where BNZ- γ treated animals showed significant regrowth of their fur (results shown in fig. 10A). To support clinical observations, BNZ- γ caused a statistically significant decrease in the levels of circulating inflammatory cytokines (IL-6 and IFN γ) back to/near the normal physiological range of NSG mice upon completion of the twice weekly BNZ- γ dosing regimen over a two week treatment duration (see figure 10B).

Example 18-BNZ-gamma, anti-IL-2 antibody, anti-IL-15 antibody, and combination of anti-IL-2 and anti-IL-15 antibodies Comparison of human-derived NSG mouse survival, immune-mediated alopecia, and cytokine levels

In this experiment, NSG mice were transplanted with 2 million hupmc on study day 0, and treatment was initiated 35 days after transplantation. Mice were treated twice weekly with IV injections of PBS control (n ═ 5), BNZ- γ at 2mg/kg (n ═ 5), anti-IL-2 antibody at 5mg/kg (n ═ 3), anti-IL-15 antibody at 5mg/kg (n ═ 3), or a combination of anti-IL-2 and anti-IL-15 antibodies each at 5mg/kg (n ═ 3), beginning on day 35 for 4 weeks. PBS control mice began to die shortly after treatment began, whereas single-treated animals began to die after treatment ceased, which was not statistically different from untreated controls (p > 0.05). The combination of anti-IL-2 and anti-IL-15 antibodies was significantly more effective compared to single antibody treatment (p ═ 0.014), with survival benefit lasting weeks after cessation of treatment, but less effective than BNZ- γ (p ═ 0.001) (results shown in fig. 11A).

Mice had significant hair loss at the beginning of treatment on day 35 post-transplantation. At about two weeks after completion of treatment (about day 63), there was a significant improvement in hair regrowth in animals treated with anti-IL-15 antibody, which appeared to be more effective than anti-IL-2 antibody. The combined antibody treatment did not appear significantly different for hair regrowth compared to the anti-IL-15 antibody alone. However, BNZ- γ treated mice appeared to have the greatest degree of hair regrowth in all 4 treatment groups, suggesting that blocking IL-9 may be important to achieve the greatest therapeutic response. (the results are shown in 11B).

The levels of IL-6 and IFN γ were also measured in this experiment. Both inflammatory cytokines showed significant elevation in PBS control NSG mice. All 4 active treatments reduced the level of each cytokine to varying degrees, with BNZ- γ and the combination antibody being the most effective. These data are consistent with previous reports that IFN γ is a downstream cytokine regulated by IL-15, where IL-15 blockade turns off IFN γ expression (Fehniger et al, 2000J. Immunol.164: 1643-7). Cytokine levels were determined using sera collected on day 50, except one animal in the anti-IL-2 antibody treated group (collected on day 45), one mouse in the PBS control untreated group (collected on day 45), and two mice in the PBS control untreated group (collected on day 40), to ensure that blood samples were collected before each animal was euthanized (results shown in figure 11C).

Example 19 immunohistochemistry of skin tissue from humanized NSG mice treated with BNZ-gamma

To characterize the nature of the skin tissue and the immune challenge around the hair follicle, immunohistochemical studies were performed on the skin tissue of humanized NSG mice for 3 weeks (before BNZ- γ) and 7 weeks (with or without BNZ- γ treatment) after 2 million Hupbmc transplants. Tissues were fixed in 4% formalin (Sigma) for 24 hours, then transferred to 70% ethanol for at least 24 hours, and then processed. The tissues were then embedded in paraffin, washed twice in 70%, 90% and 100% ethanol for two hours each, then washed twice in xylene for two hours each, and infiltrated twice with melted paraplast plus at 60C for two hours. Paraffin embedded tissues were stored at room temperature, then sectioned and stained. Anti-human CD8 antibody (BioCare Medical CRM 311C) or isotype control was used for tissue staining based on standard procedures for IHC.

It was observed that human CD 8T cells flowed into the skin tissue of the humanized NSG mice 3 weeks after transplantation. CD 8T cells remained at comparable levels 7 weeks post-transplantation in the absence of BNZ-gamma treatment. However, a significant reduction in the number of infiltrating CD8 cells was observed 7 weeks after transplantation with BNZ- γ treatment. The data are shown in figure 12.

Example 20-method of treating alopecia in human patients by administering a therapeutic Compound

Identifying a human patient having alopecia (alopecia areata, alopecia totalis, alopecia subtotalis, alopecia universalis, diffuse alopecia, alopecia areata). An effective dose of a therapeutic compound, such as an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, a tailored derivative yc-antagonistic peptide, such as a composite peptide comprising a BNZ- γ sequence or a derivative thereof, or a combination of said therapeutic compounds, as determined by a physician, is administered to the patient for a time period determined by the physician. Treatment is determined to be effective if the patient's symptoms improve or the progression of the disease has stopped or slowed.

Example 21 method of treating vitiligo in human patients by administering a therapeutic Compound

Identifying human patients with vitiligo (vitiligo and rosacea). An effective dose of a therapeutic compound, such as an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, a tailored derivative yc-antagonistic peptide, such as a composite peptide comprising a BNZ- γ sequence or a derivative thereof, or a combination of said therapeutic compounds, as determined by a physician, is administered to the patient for a time period determined by the physician. Treatment is determined to be effective if the patient's symptoms improve or the progression of the disease has stopped or slowed.

Example 22-method of treating psoriasis in a human patient by administering a therapeutic Compound

Identifying a human patient having psoriasis (psoriasis, psoriasis vulgaris, scalp psoriasis, guttate psoriasis, reverse psoriasis, psoriatic arthritis). An effective dose of a therapeutic compound, such as an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, a tailored derivative yc-antagonistic peptide, such as a composite peptide comprising a BNZ- γ sequence or a derivative thereof, or a combination of said therapeutic compounds, as determined by a physician, is administered to the patient for a time period determined by the physician. Treatment is determined to be effective if the patient's symptoms improve or the progression of the disease has stopped or slowed.

Example 23-method of treating pemphigus in human patients by administering therapeutic Compounds

Identifying a human patient having pemphigus (pemphigus, pemphigus vulgaris, pemphigus foliaceus, pemphigus proliferative, pemphigus erythematous). An effective dose of a therapeutic compound, such as an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, a tailored derivative yc-antagonistic peptide, such as a composite peptide comprising a BNZ- γ sequence or a derivative thereof, or a combination of said therapeutic compounds, as determined by a physician, is administered to the patient for a time period determined by the physician. Treatment is determined to be effective if the patient's symptoms improve or the progression of the disease has stopped or slowed.

Example 24-method of treating pemphigoid in human patients by administering therapeutic Compounds

Identifying a human patient having pemphigoid (mucosal pemphigoid, scar mucosal pemphigoid, bullous pemphigoid). An effective dose of a therapeutic compound, such as an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, a tailored derivative yc-antagonistic peptide, such as a composite peptide comprising a BNZ- γ sequence or a derivative thereof, or a combination of said therapeutic compounds, as determined by a physician, is administered to the patient for a time period determined by the physician. Treatment is determined to be effective if the patient's symptoms improve or the progression of the disease has stopped or slowed.

Example 25 method of treating GvHD in human patients by administering a therapeutic Compound

Identifying a human patient having GvHD. An effective dose of a therapeutic compound, such as an anti-CD 8 antibody, an anti-IL-2 antibody, an anti-IL-15 antibody, an anti-NKG 2A antibody, a tailored derivative yc-antagonistic peptide, such as a composite peptide comprising a BNZ- γ sequence or a derivative thereof, or a combination of said therapeutic compounds, as determined by a physician, is administered to the patient for a time period determined by the physician. Treatment is determined to be effective if the patient's symptoms improve or the progression of the disease has stopped or slowed.

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Sequence listing

<110> Biaonis LLC (BIONIZ, LLC)

<120> Effect of modulating gamma C-cytokine signaling on treating alopecia and alopecia-related disorders

<130> BION.012WO

<150> US 62/842,846

<151> 2019-05-03

<160> 46

<170> PatentIn version 3.5

<210> 1

<211> 19

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<400> 1

Ile Lys Glu Phe Leu Gln Arg Phe Ile His Ile Val Gln Ser Ile Ile

1 5 10 15

Asn Thr Ser

<210> 2

<211> 11

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X = D or E

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X = E or Q or N

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X = S or R

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X = I or K

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X = L or I

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X = any amino acid

<400> 2

Xaa Phe Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln

1 5 10

<210> 3

<211> 19

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<400> 3

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

1 5 10 15

Asn Thr Ser

<210> 4

<211> 20

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<400> 4

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

1 5 10 15

Ser Thr Leu Thr

20

<210> 5

<211> 21

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<400> 5

Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile

1 5 10 15

His Gln His Leu Ser

20

<210> 6

<211> 21

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<400> 6

Leu Glu Asn Phe Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr

1 5 10 15

Ser Lys Cys Ser Ser

20

<210> 7

<211> 21

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<400> 7

Ala Leu Thr Phe Leu Glu Ser Leu Leu Glu Leu Phe Gln Lys Glu Lys

1 5 10 15

Met Arg Gly Met Arg

20

<210> 8

<211> 20

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<400> 8

Asp Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp

1 5 10 15

Asn Lys Ile Leu

20

<210> 9

<211> 19

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X = D or E

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X = E or Q or N or polar amino acids

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X = S or R or polar amino acid

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X = non-polar amino acid

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X = I or K or a non-polar amino acid

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X = L or I or aliphatic amino acids

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X = non-polar amino acid

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> X = charged amino acid

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> X = I or K

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (19)..(19)

<223> X = any amino acid

<400> 9

Xaa Xaa Xaa Phe Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa

<210> 10

<211> 19

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X = D or E

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X = E or Q or N or polar amino acids

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X = S or R or polar amino acid

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X = non-polar amino acid

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X = I or K or a non-polar amino acid

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X = L or I or aliphatic amino acids

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X = non-polar amino acid

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> X = charged amino acid

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> X = any amino acid

<400> 10

Xaa Xaa Xaa Phe Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Xaa Xaa Ile

1 5 10 15

Xaa Thr Ser

<210> 11

<211> 20

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> S-pentenylalanine; to the S-pentenylalanine in position 5

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> S-pentenylalanine; to the S-pentenylalanine in position 1

<400> 11

Ala Ile Lys Glu Ala Leu Gln Arg Phe Ile His Ile Val Gln Ser Ile

1 5 10 15

Ile Asn Thr Ser

20

<210> 12

<211> 19

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> S-pentenylalanine; to S-pentenylalanine at position 12

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> S-pentenylalanine; to S-pentenylalanine in position 8

<400> 12

Ile Lys Glu Phe Leu Gln Arg Ala Ile His Ile Ala Gln Ser Ile Ile

1 5 10 15

Asn Thr Ser

<210> 13

<211> 19

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> R-octenylalanine; to S-pentenylalanine in position 15

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> S-pentenylalanine; to the R-octenylalanine in position 8

<400> 13

Ile Lys Glu Phe Leu Gln Arg Ala Ile His Ile Val Gln Ser Ala Ile

1 5 10 15

Asn Thr Ser

<210> 14

<211> 19

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> S-pentenylalanine; to S-pentenylalanine at position 16

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> S-pentenylalanine; to S-pentenylalanine at position 12

<400> 14

Ile Lys Glu Phe Leu Gln Arg Phe Ile His Ile Ala Gln Ser Ile Ala

1 5 10 15

Asn Thr Ser

<210> 15

<211> 19

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> R-octenylalanine; to S-pentenylalanine at position 19

<220>

<221> MISC_FEATURE

<222> (19)..(19)

<223> S-pentenylalanine; to the R-octenylalanine in position 12

<400> 15

Ile Lys Glu Phe Leu Gln Arg Phe Ile His Ile Ala Gln Ser Ile Ile

1 5 10 15

Asn Thr Ala

<210> 16

<211> 20

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> S-pentenylalanine; to the S-pentenylalanine in position 5

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> S-pentenylalanine; to the S-pentenylalanine in position 1

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> S-pentenylalanine; to the S-pentenylalanine at position 13

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> S-pentenylalanine; to S-pentenylalanine in position 9

<400> 16

Ala Ile Lys Glu Ala Leu Gln Arg Ala Ile His Ile Ala Gln Ser Ile

1 5 10 15

Ile Asn Thr Ser

20

<210> 17

<211> 20

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> S-pentenylalanine; to the S-pentenylalanine in position 5

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> S-pentenylalanine; to the S-pentenylalanine in position 1

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> R-octenylalanine; to S-pentenylalanine at position 16

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> S-pentenylalanine; to the R-octenylalanine in position 8

<400> 17

Ala Ile Lys Glu Ala Leu Gln Arg Ala Ile His Ile Val Gln Ser Ala

1 5 10 15

Ile Asn Thr Ser

20

<210> 18

<211> 20

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> S-pentenylalanine; to the S-pentenylalanine in position 5

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> S-pentenylalanine; to the S-pentenylalanine in position 1

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> S-pentenylalanine; to S-pentenylalanine at position 17

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> S-pentenylalanine; to the S-pentenylalanine at position 13

<400> 18

Ala Ile Lys Glu Ala Leu Gln Arg Phe Ile His Ile Ala Gln Ser Ile

1 5 10 15

Ala Asn Thr Ser

20

<210> 19

<211> 20

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> S-pentenylalanine; to the S-pentenylalanine in position 5

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> S-pentenylalanine; to the S-pentenylalanine in position 1

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> R-octenylalanine; to the S-pentenylalanine in position 20

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> S-pentenylalanine; to the R-octenylalanine at position 13

<400> 19

Ala Ile Lys Glu Ala Leu Gln Arg Phe Ile His Ile Ala Gln Ser Ile

1 5 10 15

Ile Asn Thr Ala

20

<210> 20

<211> 20

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> S-pentenylalanine; to the S-pentenylalanine in position 5

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> S-pentenylalanine; to the S-pentenylalanine in position 1

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> S-pentenylalanine; to the S-pentenylalanine at position 13

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> S-pentenylalanine; to S-pentenylalanine in position 9

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> stereochemical configuration of D

<220>

<221> MISC_FEATURE

<222> (19)..(19)

<223> stereochemical configuration of D

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> stereochemical configuration of D

<400> 20

Ala Ile Lys Glu Ala Leu Gln Arg Ala Ile His Ile Ala Gln Ser Ile

1 5 10 15

Ile Asn Thr Ser

20

<210> 21

<211> 20

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> S-pentenylalanine; to the S-pentenylalanine in position 5

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> S-pentenylalanine; to the S-pentenylalanine in position 1

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> R-octenylalanine; to S-pentenylalanine at position 16

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> S-pentenylalanine; to the R-octenylalanine in position 9

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> stereochemical configuration of D

<220>

<221> MISC_FEATURE

<222> (19)..(19)

<223> stereochemical configuration of D

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> stereochemical configuration of D

<400> 21

Ala Ile Lys Glu Ala Leu Gln Arg Ala Ile His Ile Val Gln Ser Ala

1 5 10 15

Ile Asn Thr Ser

20

<210> 22

<211> 20

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> S-pentenylalanine; to the S-pentenylalanine in position 5

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> S-pentenylalanine; to the S-pentenylalanine in position 1

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> S-pentenylalanine; to S-pentenylalanine at position 17

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> S-pentenylalanine; to the S-pentenylalanine at position 13

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> stereochemical configuration of D

<220>

<221> MISC_FEATURE

<222> (19)..(19)

<223> stereochemical configuration of D

<220>

<221> MISC_FEATURE

<222> (20)..(20)

<223> stereochemical configuration of D

<400> 22

Ala Ile Lys Glu Ala Leu Gln Arg Phe Ile His Ile Ala Gln Ser Ile

1 5 10 15

Ala Asn Thr Ser

20

<210> 23

<211> 10

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> S-pentenylalanine; to S-pentenylalanine in position 8

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> S-pentenylalanine; to the S-pentenylalanine in position 4

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X = any amino acid

<400> 23

Xaa Xaa Xaa Ala Xaa Xaa Xaa Ala Xaa Xaa

1 5 10

<210> 24

<211> 11

<212> PRT

<213> Artificial (Artificial)

<220>

<223> synthetic

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> R-octenylalanine; to the S-pentenylalanine in position 10

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X = any amino acid

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> S-pentenylalanine; to the R-octenylalanine in position 3

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X = any amino acid

<400> 24

Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa

1 5 10

<210> 25

<211> 708

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 25

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgagccagt tccgggtgtc gccgctggat cggacctgga acctgggcga gacagtggag 120

ctgaagtgcc aggtgctgct gtccaacccg acgtcgggct gctcgtggct cttccagccg 180

cgcggcgccg ccgccagtcc caccttcctc ctatacctct cccaaaacaa gcccaaggcg 240

gccgaggggc tggacaccca gcggttctcg ggcaagaggt tgggggacac cttcgtcctc 300

accctgagcg acttccgccg agagaacgag ggctactatt tctgctcggc cctgagcaac 360

tccatcatgt acttcagcca cttcgtgccg gtcttcctgc cagcgaagcc caccacgacg 420

ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 480

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 540

tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 600

gttatcaccc tttactgcaa ccacaggaac cgaagacgtg tttgcaaatg tccccggcct 660

gtggtcaaat cgggagacaa gcccagcctt tcggcgagat acgtctaa 708

<210> 26

<211> 235

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 26

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr

20 25 30

Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser

35 40 45

Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala

50 55 60

Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala

65 70 75 80

Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp

85 90 95

Thr Phe Val Leu Thr Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr

100 105 110

Tyr Phe Cys Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe

115 120 125

Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg

130 135 140

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

145 150 155 160

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

165 170 175

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

180 185 190

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His

195 200 205

Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser

210 215 220

Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val

225 230 235

<210> 27

<211> 732

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 27

atgcggccgc ggctgtggct cctcttggcc gcgcagctga cagttctcca tggcaactca 60

gtcctccagc agacccctgc atacataaag gtgcaaacca acaagatggt gatgctgtcc 120

tgcgaggcta aaatctccct cagtaacatg cgcatctact ggctgagaca gcgccaggca 180

ccgagcagtg acagtcacca cgagttcctg gccctctggg attccgcaaa agggactatc 240

cacggtgaag aggtggaaca ggagaagata gctgtgtttc gggatgcaag ccggttcatt 300

ctcaatctca caagcgtgaa gccggaagac agtggcatct acttctgcat gatcgtcggg 360

agccccgagc tgaccttcgg gaagggaact cagctgagtg tggttgattt ccttcccacc 420

actgcccagc ccaccaagaa gtccaccctc aagaagagag tgtgccggtt acccaggcca 480

gagacccaga agggcccact ttgtagcccc atcacccttg gcctgctggt ggctggcgtc 540

ctggttctgc tggtttccct gggagtggcc atccacctgt gctgccggcg gaggagagcc 600

cggcttcgtt tcatgaaaca gcctcaaggg gaaggtatat caggaacctt tgtcccccaa 660

tgcctgcatg gatactacag caatactaca acctcacaga agctgcttaa cccatggatc 720

ctgaaaacat ag 732

<210> 28

<211> 243

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 28

Met Arg Pro Arg Leu Trp Leu Leu Leu Ala Ala Gln Leu Thr Val Leu

1 5 10 15

His Gly Asn Ser Val Leu Gln Gln Thr Pro Ala Tyr Ile Lys Val Gln

20 25 30

Thr Asn Lys Met Val Met Leu Ser Cys Glu Ala Lys Ile Ser Leu Ser

35 40 45

Asn Met Arg Ile Tyr Trp Leu Arg Gln Arg Gln Ala Pro Ser Ser Asp

50 55 60

Ser His His Glu Phe Leu Ala Leu Trp Asp Ser Ala Lys Gly Thr Ile

65 70 75 80

His Gly Glu Glu Val Glu Gln Glu Lys Ile Ala Val Phe Arg Asp Ala

85 90 95

Ser Arg Phe Ile Leu Asn Leu Thr Ser Val Lys Pro Glu Asp Ser Gly

100 105 110

Ile Tyr Phe Cys Met Ile Val Gly Ser Pro Glu Leu Thr Phe Gly Lys

115 120 125

Gly Thr Gln Leu Ser Val Val Asp Phe Leu Pro Thr Thr Ala Gln Pro

130 135 140

Thr Lys Lys Ser Thr Leu Lys Lys Arg Val Cys Arg Leu Pro Arg Pro

145 150 155 160

Glu Thr Gln Lys Gly Pro Leu Cys Ser Pro Ile Thr Leu Gly Leu Leu

165 170 175

Val Ala Gly Val Leu Val Leu Leu Val Ser Leu Gly Val Ala Ile His

180 185 190

Leu Cys Cys Arg Arg Arg Arg Ala Arg Leu Arg Phe Met Lys Gln Pro

195 200 205

Gln Gly Glu Gly Ile Ser Gly Thr Phe Val Pro Gln Cys Leu His Gly

210 215 220

Tyr Tyr Ser Asn Thr Thr Thr Ser Gln Lys Leu Leu Asn Pro Trp Ile

225 230 235 240

Leu Lys Thr

<210> 29

<211> 462

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 29

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt 60

gcacctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 120

ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 180

acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 240

gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 300

agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 360

acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 420

tggattacct tttgtcaaag catcatctca acactgactt ga 462

<210> 30

<211> 153

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 30

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

35 40 45

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe

50 55 60

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

85 90 95

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

100 105 110

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

115 120 125

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

130 135 140

Cys Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210> 31

<211> 489

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 31

atgagaattt cgaaaccaca tttgagaagt atttccatcc agtgctactt gtgtttactt 60

ctaaacagtc attttctaac tgaagctggc attcatgtct tcattttggg ctgtttcagt 120

gcagggcttc ctaaaacaga agccaactgg gtgaatgtaa taagtgattt gaaaaaaatt 180

gaagatctta ttcaatctat gcatattgat gctactttat atacggaaag tgatgttcac 240

cccagttgca aagtaacagc aatgaagtgc tttctcttgg agttacaagt tatttcactt 300

gagtccggag atgcaagtat tcatgataca gtagaaaatc tgatcatcct agcaaacaac 360

agtttgtctt ctaatgggaa tgtaacagaa tctggatgca aagaatgtga ggaactggag 420

gaaaaaaata ttaaagaatt tttgcagagt tttgtacata ttgtccaaat gttcatcaac 480

acttcttga 489

<210> 32

<211> 162

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 32

Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr

1 5 10 15

Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His

20 25 30

Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala

35 40 45

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

50 55 60

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

65 70 75 80

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

85 90 95

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

100 105 110

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

115 120 125

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

130 135 140

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

145 150 155 160

Thr Ser

<210> 33

<211> 702

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 33

atggataacc aaggagtaat ctactcagac ctgaatctgc ccccaaaccc aaagaggcag 60

caacgaaaac ctaaaggcaa taaaagctcc attttagcaa ctgaacagga aataacctat 120

gcggaattaa accttcaaaa agcttctcag gattttcaag ggaatgacaa aacctatcac 180

tgcaaagatt taccatcagc tccagagaag ctcattgttg ggatcctggg aattatctgt 240

cttatcttaa tggcctctgt ggtaacgata gttgttattc cctctacatt aatacagagg 300

cacaacaatt cttccctgaa tacaagaact cagaaagcac gtcattgtgg ccattgtcct 360

gaggagtgga ttacatattc caacagttgt tactacattg gtaaggaaag aagaacttgg 420

gaagagagtt tgctggcctg tacttcgaag aactccagtc tgctttctat agataatgaa 480

gaagaaatga aatttctgtc catcatttca ccatcctcat ggattggtgt gtttcgtaac 540

agcagtcatc atccatgggt gacaatgaat ggtttggctt tcaaacatga gataaaagac 600

tcagataatg ctgaacttaa ctgtgcagtg ctacaagtaa atcgacttaa atcagcccag 660

tgtggatctt caataatata tcattgtaag cataagcttt ag 702

<210> 34

<211> 233

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 34

Met Asp Asn Gln Gly Val Ile Tyr Ser Asp Leu Asn Leu Pro Pro Asn

1 5 10 15

Pro Lys Arg Gln Gln Arg Lys Pro Lys Gly Asn Lys Ser Ser Ile Leu

20 25 30

Ala Thr Glu Gln Glu Ile Thr Tyr Ala Glu Leu Asn Leu Gln Lys Ala

35 40 45

Ser Gln Asp Phe Gln Gly Asn Asp Lys Thr Tyr His Cys Lys Asp Leu

50 55 60

Pro Ser Ala Pro Glu Lys Leu Ile Val Gly Ile Leu Gly Ile Ile Cys

65 70 75 80

Leu Ile Leu Met Ala Ser Val Val Thr Ile Val Val Ile Pro Ser Thr

85 90 95

Leu Ile Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gln Lys

100 105 110

Ala Arg His Cys Gly His Cys Pro Glu Glu Trp Ile Thr Tyr Ser Asn

115 120 125

Ser Cys Tyr Tyr Ile Gly Lys Glu Arg Arg Thr Trp Glu Glu Ser Leu

130 135 140

Leu Ala Cys Thr Ser Lys Asn Ser Ser Leu Leu Ser Ile Asp Asn Glu

145 150 155 160

Glu Glu Met Lys Phe Leu Ser Ile Ile Ser Pro Ser Ser Trp Ile Gly

165 170 175

Val Phe Arg Asn Ser Ser His His Pro Trp Val Thr Met Asn Gly Leu

180 185 190

Ala Phe Lys His Glu Ile Lys Asp Ser Asp Asn Ala Glu Leu Asn Cys

195 200 205

Ala Val Leu Gln Val Asn Arg Leu Lys Ser Ala Gln Cys Gly Ser Ser

210 215 220

Ile Ile Tyr His Cys Lys His Lys Leu

225 230

<210> 35

<211> 648

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 35

atggataacc aaggagtaat ctactcagac ctgaatctgc ccccaaaccc aaagaggcag 60

caacgaaaac ctaaaggcaa taaaagctcc attttagcaa ctgaacagga aataacctat 120

gcggaattaa accttcaaaa agcttctcag gattttcaag ggaatgacaa aacctatcac 180

tgcaaagatt taccatcagc tccagagaag ctcattgttg ggatcctggg aattatctgt 240

cttatcttaa tggcctctgt ggtaacgata gttgttattc cctcacgtca ttgtggccat 300

tgtcctgagg agtggattac atattccaac agttgttact acattggtaa ggaaagaaga 360

acttgggaag agagtttgct ggcctgtact tcgaagaact ccagtctgct ttctatagat 420

aatgaagaag aaatgaaatt tctgtccatc atttcaccat cctcatggat tggtgtgttt 480

cgtaacagca gtcatcatcc atgggtgaca atgaatggtt tggctttcaa acatgagata 540

aaagactcag ataatgctga acttaactgt gcagtgctac aagtaaatcg acttaaatca 600

gcccagtgtg gatcttcaat aatatatcat tgtaagcata agctttag 648

<210> 36

<211> 215

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 36

Met Asp Asn Gln Gly Val Ile Tyr Ser Asp Leu Asn Leu Pro Pro Asn

1 5 10 15

Pro Lys Arg Gln Gln Arg Lys Pro Lys Gly Asn Lys Ser Ser Ile Leu

20 25 30

Ala Thr Glu Gln Glu Ile Thr Tyr Ala Glu Leu Asn Leu Gln Lys Ala

35 40 45

Ser Gln Asp Phe Gln Gly Asn Asp Lys Thr Tyr His Cys Lys Asp Leu

50 55 60

Pro Ser Ala Pro Glu Lys Leu Ile Val Gly Ile Leu Gly Ile Ile Cys

65 70 75 80

Leu Ile Leu Met Ala Ser Val Val Thr Ile Val Val Ile Pro Ser Arg

85 90 95

His Cys Gly His Cys Pro Glu Glu Trp Ile Thr Tyr Ser Asn Ser Cys

100 105 110

Tyr Tyr Ile Gly Lys Glu Arg Arg Thr Trp Glu Glu Ser Leu Leu Ala

115 120 125

Cys Thr Ser Lys Asn Ser Ser Leu Leu Ser Ile Asp Asn Glu Glu Glu

130 135 140

Met Lys Phe Leu Ser Ile Ile Ser Pro Ser Ser Trp Ile Gly Val Phe

145 150 155 160

Arg Asn Ser Ser His His Pro Trp Val Thr Met Asn Gly Leu Ala Phe

165 170 175

Lys His Glu Ile Lys Asp Ser Asp Asn Ala Glu Leu Asn Cys Ala Val

180 185 190

Leu Gln Val Asn Arg Leu Lys Ser Ala Gln Cys Gly Ser Ser Ile Ile

195 200 205

Tyr His Cys Lys His Lys Leu

210 215

<210> 37

<211> 696

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 37

atgaataaac aaagaggaac cttctcagaa gtgagtctgg cccaggaccc aaagcggcag 60

caaaggaaac ctaaaggcaa taaaagctcc atttcaggaa ccgaacagga aatattccaa 120

gtagaattaa atcttcaaaa tccttccctg aatcatcaag ggattgataa aatatatgac 180

tgccaaggtt tactgccacc tccagagaag ctcactgccg aggtcctagg aatcatttgc 240

attgtcctga tggccactgt gttaaaaaca atagttctta ttcctttcct ggagcagaac 300

aatttttccc cgaatacaag aacgcagaaa gcacgtcatt gtggccattg tcctgaggag 360

tggattacat attccaacag ttgttattac attggtaagg aaagaagaac ttgggaagag 420

agtttgctgg cctgtacttc gaagaactcc agtctgcttt ctatagataa tgaagaagaa 480

atgaaatttc tggccagcat tttaccttcc tcatggattg gtgtgtttcg taacagcagt 540

catcatccat gggtgacaat aaatggtttg gctttcaaac ataagataaa agactcagat 600

aatgctgaac ttaactgtgc agtgctacaa gtaaatcgac ttaaatcagc ccagtgtgga 660

tcttcaatga tatatcattg taagcataag ctttag 696

<210> 38

<211> 231

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 38

Met Asn Lys Gln Arg Gly Thr Phe Ser Glu Val Ser Leu Ala Gln Asp

1 5 10 15

Pro Lys Arg Gln Gln Arg Lys Pro Lys Gly Asn Lys Ser Ser Ile Ser

20 25 30

Gly Thr Glu Gln Glu Ile Phe Gln Val Glu Leu Asn Leu Gln Asn Pro

35 40 45

Ser Leu Asn His Gln Gly Ile Asp Lys Ile Tyr Asp Cys Gln Gly Leu

50 55 60

Leu Pro Pro Pro Glu Lys Leu Thr Ala Glu Val Leu Gly Ile Ile Cys

65 70 75 80

Ile Val Leu Met Ala Thr Val Leu Lys Thr Ile Val Leu Ile Pro Phe

85 90 95

Leu Glu Gln Asn Asn Phe Ser Pro Asn Thr Arg Thr Gln Lys Ala Arg

100 105 110

His Cys Gly His Cys Pro Glu Glu Trp Ile Thr Tyr Ser Asn Ser Cys

115 120 125

Tyr Tyr Ile Gly Lys Glu Arg Arg Thr Trp Glu Glu Ser Leu Leu Ala

130 135 140

Cys Thr Ser Lys Asn Ser Ser Leu Leu Ser Ile Asp Asn Glu Glu Glu

145 150 155 160

Met Lys Phe Leu Ala Ser Ile Leu Pro Ser Ser Trp Ile Gly Val Phe

165 170 175

Arg Asn Ser Ser His His Pro Trp Val Thr Ile Asn Gly Leu Ala Phe

180 185 190

Lys His Lys Ile Lys Asp Ser Asp Asn Ala Glu Leu Asn Cys Ala Val

195 200 205

Leu Gln Val Asn Arg Leu Lys Ser Ala Gln Cys Gly Ser Ser Met Ile

210 215 220

Tyr His Cys Lys His Lys Leu

225 230

<210> 39

<211> 651

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 39

atggggtgga ttcgtggtcg gaggtctcga cacagctggg agatgagtga atttcataat 60

tataacttgg atctgaagaa gagtgatttt tcaacacgat ggcaaaagca aagatgtcca 120

gtagtcaaaa gcaaatgtag agaaaatgca tctccatttt ttttctgctg cttcatcgct 180

gtagccatgg gaatccgttt cattattatg gtaacaatat ggagtgctgt attcctaaac 240

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 300

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 360

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 420

gaggaccagg atttacttaa actggtgaag tcatatcatt ggatgggact agtacacatt 480

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 540

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 600

gaaaactgtt caactccaaa tacgtacatc tgcatgcaaa ggactgtgta a 651

<210> 40

<211> 216

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 40

Met Gly Trp Ile Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser

1 5 10 15

Glu Phe His Asn Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr

20 25 30

Arg Trp Gln Lys Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu

35 40 45

Asn Ala Ser Pro Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly

50 55 60

Ile Arg Phe Ile Ile Met Val Thr Ile Trp Ser Ala Val Phe Leu Asn

65 70 75 80

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

85 90 95

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

100 105 110

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

115 120 125

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

130 135 140

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

145 150 155 160

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

165 170 175

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

180 185 190

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

195 200 205

Tyr Ile Cys Met Gln Arg Thr Val

210 215

<210> 41

<211> 723

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 41

atgagtaaac aaagaggaac cttctcagaa gtgagtctgg cccaggaccc aaagtggcag 60

caaaggaaac ctaaaggcaa taaaagctcc atttcaggaa ccgaacagga aatattccaa 120

gtagaattaa accttcaaaa tgcttctctg aatcatcaag ggattgataa aatatatgac 180

tgccaaggtt tactgccacc tccagaaaag ctcactgccg aggtcctagg aatcatttgc 240

attgtcctga tggccactgt gttaaaaaca atagttctta ttcctttcct ggagcagaac 300

aattcttccc cgaatgcaag aacccagaaa gcacgtcatt gtggccattg tcctgaggag 360

tggattacat attccaacag ttgttattac attggtaagg aaagaagaac ttgggaagag 420

agtttgcagg cctgtgcttc aaagaactct tctagtctgc tttgtataga taatgaagaa 480

gaaatgaaat ttctggccag cattttacct tcctcatgga ttggtgtgtt tcgtaacagc 540

agtcatcatc catgggtgac aataaatggt ttggctttca aacatgagat aaaagactca 600

gatcatgctg aacgtaactg tgcaatgcta catgtacgtg gacttatatc agaccagtgt 660

ggatcttcaa gaatcattag acggggtttc atcatgttga ccaggctggt cttgaactcc 720

tga 723

<210> 42

<211> 240

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 42

Met Ser Lys Gln Arg Gly Thr Phe Ser Glu Val Ser Leu Ala Gln Asp

1 5 10 15

Pro Lys Trp Gln Gln Arg Lys Pro Lys Gly Asn Lys Ser Ser Ile Ser

20 25 30

Gly Thr Glu Gln Glu Ile Phe Gln Val Glu Leu Asn Leu Gln Asn Ala

35 40 45

Ser Leu Asn His Gln Gly Ile Asp Lys Ile Tyr Asp Cys Gln Gly Leu

50 55 60

Leu Pro Pro Pro Glu Lys Leu Thr Ala Glu Val Leu Gly Ile Ile Cys

65 70 75 80

Ile Val Leu Met Ala Thr Val Leu Lys Thr Ile Val Leu Ile Pro Phe

85 90 95

Leu Glu Gln Asn Asn Ser Ser Pro Asn Ala Arg Thr Gln Lys Ala Arg

100 105 110

His Cys Gly His Cys Pro Glu Glu Trp Ile Thr Tyr Ser Asn Ser Cys

115 120 125

Tyr Tyr Ile Gly Lys Glu Arg Arg Thr Trp Glu Glu Ser Leu Gln Ala

130 135 140

Cys Ala Ser Lys Asn Ser Ser Ser Leu Leu Cys Ile Asp Asn Glu Glu

145 150 155 160

Glu Met Lys Phe Leu Ala Ser Ile Leu Pro Ser Ser Trp Ile Gly Val

165 170 175

Phe Arg Asn Ser Ser His His Pro Trp Val Thr Ile Asn Gly Leu Ala

180 185 190

Phe Lys His Glu Ile Lys Asp Ser Asp His Ala Glu Arg Asn Cys Ala

195 200 205

Met Leu His Val Arg Gly Leu Ile Ser Asp Gln Cys Gly Ser Ser Arg

210 215 220

Ile Ile Arg Arg Gly Phe Ile Met Leu Thr Arg Leu Val Leu Asn Ser

225 230 235 240

<210> 43

<211> 477

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 43

atgaataaac aaagaggaac ctactcagaa gtgagtctgg cccaggaccc aaagaggcag 60

caaaggaaac ttaagggcaa taaaatctcc atttcaggaa ccaaacagga aatattccaa 120

gtagaattaa accttcaaaa tgcttcttcg gatcatcaag ggaatgacaa gacatatcac 180

tgcaaaggtt tactgccacc tccagagaag ctcactgctg aggtcctagg aatcatttgc 240

attgtcctga tggccactgt gttaaaaaca atagttctta ttccttgtat tggagtactg 300

gagcagaaca atttttccct gaatagaaga atgcagaaag cacgtcattg tggccattgt 360

cctgaggagt ggattacata ttccaacagt tgttattaca ttggtaagga aagaagaact 420

tgggaagaaa gagtttgctg gcctgtgctt cgaagaactc tgatctgctt tctatag 477

<210> 44

<211> 158

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 44

Met Asn Lys Gln Arg Gly Thr Tyr Ser Glu Val Ser Leu Ala Gln Asp

1 5 10 15

Pro Lys Arg Gln Gln Arg Lys Leu Lys Gly Asn Lys Ile Ser Ile Ser

20 25 30

Gly Thr Lys Gln Glu Ile Phe Gln Val Glu Leu Asn Leu Gln Asn Ala

35 40 45

Ser Ser Asp His Gln Gly Asn Asp Lys Thr Tyr His Cys Lys Gly Leu

50 55 60

Leu Pro Pro Pro Glu Lys Leu Thr Ala Glu Val Leu Gly Ile Ile Cys

65 70 75 80

Ile Val Leu Met Ala Thr Val Leu Lys Thr Ile Val Leu Ile Pro Cys

85 90 95

Ile Gly Val Leu Glu Gln Asn Asn Phe Ser Leu Asn Arg Arg Met Gln

100 105 110

Lys Ala Arg His Cys Gly His Cys Pro Glu Glu Trp Ile Thr Tyr Ser

115 120 125

Asn Ser Cys Tyr Tyr Ile Gly Lys Glu Arg Arg Thr Trp Glu Glu Arg

130 135 140

Val Cys Trp Pro Val Leu Arg Arg Thr Leu Ile Cys Phe Leu

145 150 155

<210> 45

<211> 774

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 45

atgagtaaac aaagaggaac cttctcagaa gtgagtctgg cccaggaccc aaagtggcag 60

caaaggaaac ctaaaggcaa taaaagctcc atttcaggaa ccgaacagga aatattccaa 120

gtagaattaa accttcaaaa tgcttctctg aatcatcaag ggattgataa aatatatgac 180

tgccaaggtt tactgccacc tccagaaaag ctcactgccg aggtcctagg aatcatttgc 240

attgtcctga tggccactgt gttaaaaaca atagttctta ttcctttcct ggagcagaac 300

aattcttccc cgaatgcaag aacccagaaa gcacgtcatt gtggccattg tcctgaggag 360

tggattacat attccaacag ttgttattac attggtaagg aaagaagaac ttgggaagag 420

agtttgcagg cctgtgcttc aaagaactct tctagtctgc tttgtataga taatgaagaa 480

gaaatgaaat ttctggccag cattttacct tcctcatgga ttggtgtgtt tcgtaacagc 540

agtcatcatc catgggtgac aataaatggt ttggctttca aacatgagat aaaagactca 600

gatcatgctg aacgtaactg tgcaatgcta catgtacgtg gacttatatc agaccagtgt 660

ggatcttcaa gaatcattgt gagcataagc tttagaatta aagcgcttga gcttgcagtg 720

catcagataa aattttatat ttgttcaaac agaaatgata ttatgattgc ataa 774

<210> 46

<211> 257

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 46

Met Ser Lys Gln Arg Gly Thr Phe Ser Glu Val Ser Leu Ala Gln Asp

1 5 10 15

Pro Lys Trp Gln Gln Arg Lys Pro Lys Gly Asn Lys Ser Ser Ile Ser

20 25 30

Gly Thr Glu Gln Glu Ile Phe Gln Val Glu Leu Asn Leu Gln Asn Ala

35 40 45

Ser Leu Asn His Gln Gly Ile Asp Lys Ile Tyr Asp Cys Gln Gly Leu

50 55 60

Leu Pro Pro Pro Glu Lys Leu Thr Ala Glu Val Leu Gly Ile Ile Cys

65 70 75 80

Ile Val Leu Met Ala Thr Val Leu Lys Thr Ile Val Leu Ile Pro Phe

85 90 95

Leu Glu Gln Asn Asn Ser Ser Pro Asn Ala Arg Thr Gln Lys Ala Arg

100 105 110

His Cys Gly His Cys Pro Glu Glu Trp Ile Thr Tyr Ser Asn Ser Cys

115 120 125

Tyr Tyr Ile Gly Lys Glu Arg Arg Thr Trp Glu Glu Ser Leu Gln Ala

130 135 140

Cys Ala Ser Lys Asn Ser Ser Ser Leu Leu Cys Ile Asp Asn Glu Glu

145 150 155 160

Glu Met Lys Phe Leu Ala Ser Ile Leu Pro Ser Ser Trp Ile Gly Val

165 170 175

Phe Arg Asn Ser Ser His His Pro Trp Val Thr Ile Asn Gly Leu Ala

180 185 190

Phe Lys His Glu Ile Lys Asp Ser Asp His Ala Glu Arg Asn Cys Ala

195 200 205

Met Leu His Val Arg Gly Leu Ile Ser Asp Gln Cys Gly Ser Ser Arg

210 215 220

Ile Ile Val Ser Ile Ser Phe Arg Ile Lys Ala Leu Glu Leu Ala Val

225 230 235 240

His Gln Ile Lys Phe Tyr Ile Cys Ser Asn Arg Asn Asp Ile Met Ile

245 250 255

Ala