Methods of administering a composition comprising a Tn3 scaffold and a CD40L-specific monomer
The present invention provides Tenascin-3 FnIII domain-based scaffolds that specifically bind to CD40L. The invention further provides engineered variants with increased affinity for the target. The present invention is also related to engineered scaffolds as prophylactic, diagnostic, or therapeutic agents, in particular for therapeutic uses against SLE and other autoimmune diseases and conditions.
1. A method comprising administering an effective amount of a composition comprising a Tn3 scaffold to a subject with an autoimmune disease, wherein the Tn3 scaffold comprises at least one CD40L-specific monomer subunit, wherein the monomer subunit comprises seven beta strands designated A, B, C, D, E, F, and G, and six loop regions designated AB, BC, CD, DE, EF, and FG, wherein the AB loop comprises SEQ ID NO: 4, the BC loop comprises a sequence selected from the group consisting of SEQ ID NOS: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, and 168, the CD loop comprises SEQ ID NO: 6, the DE loop comprises a sequence selected from the group consisting of SEQ ID NOs: 94, 95, 96, 97, 98, and 169, the EF loop comprises SEQ ID NO: 8, and the FG loop comprises a sequence selected from the group consisting of SEQ ID NOS: 9, 99, 139, and 170.
2. The method of claim 1 , wherein the autoimmune disease is selected from the group consisting of alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Sjogren's syndrome, atherosclerosis, a retinopathy, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, Grave's disease, sepsis, arthritis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, anaphylactic shock, organ ischemia, spinal cord injury and allograft rejection, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, glomerulonephritis, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, Raynaud's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteritis giant cell arteritis, ulcerative colitis, uveitis, vasculitides, vitiligo, and Wegener's granulomatosis.
3. The method of claim 2 , wherein the autoimmune disease is systemic lupus erythematosus.
4. The method of claim 2 , wherein the autoimmune disease is arthritis.
5. The method of claim 4 , wherein the arthritis is juvenile arthritis.
6. The method of claim 4 , wherein the arthritis is rheumatoid arthritis.
7. The method of claim 2 , wherein the autoimmune disease is Sjogren's syndrome.
8. The method of claim 2 , wherein the Tn3 scaffold comprises two CD40L-specific monomer subunits connected in tandem.
9. The method of claim 8 , wherein at least one CD40L-specific monomer subunit is fused or conjugated to a heterologous moiety.
10. The method of claim 9 , wherein the heterologous moiety is a protein.
11. The method of claim 9 , wherein the heterologous moiety is an antibody.
12. The method of claim 9 , wherein the heterologous moiety is an imaging agent.
13. The method of claim 9 , wherein the heterologous moiety is a drug.
14. The method of claim 10 , wherein the protein is selected from the group consisting of: an enzyme, a binding peptide, a non-fibronectin type III domain scaffold, an epitope tag, a human serum albumin (HSA) FcRn binding portion, an albumin binding domain, and a cytokine.
15. The method of claim 11 , wherein the antibody is selected from the group consisting of: an Fc domain of an antibody, an antibody fragment, and a single chain antibody.
16. The method of claim 12 , wherein the imaging agent is a radionuclide or biotin.
17. The method of claim 13 , wherein the drug is a cytotoxic agent or a radioactive compound.
18. The method of claim 9 , wherein the heterologous moiety is selected from the group consisting of: an organic polymer, an inorganic polymer, a ligand, and a receptor.
19. The method of claim 8 , wherein at least one CD40L-specific monomer subunit is fused or conjugated to a PEG.
20. The method of claim 8 , wherein at least one CD40L-specific monomer subunit is fused or conjugated to an albumin.
21. The method of claim 20 , wherein the albumin is human serum albumin (HSA).
22. The method of claim 21 , wherein the HSA is a variant HSA.
23. The method of claim 22 , wherein the variant HSA comprises the amino acid sequence of SEQ ID NO: 133.
24. The method of claim 1 , wherein the A beta strand consists of SEQ ID NO:11, the B beta strand consists of SEQ ID NO:12, the C beta strand consists of SEQ ID NO:13 or SEQ ID NO:14, the D beta strand consists of SEQ ID NO:15, the E beta strand consists of SEQ ID NO:16, the F beta strand consists of SEQ ID NO:17, and the G beta strand consists of SEQ ID NO:18.
25. The method of claim 1 , wherein the Tn3 scaffold comprises a sequence selected from the group consisting of: SEQ ID NOs: 134, 135, 144, 145, 146, 166, 205, 206, 207 and 208.
26. The method of claim 1 , wherein:
(a) the sequence of the BC loop comprises SEQ ID NO: 83, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(b) the sequence of the BC loop comprises SEQ ID NO: 83, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 99;
(c) the sequence of the BC loop comprises SEQ ID NO: 84, the sequence of the DE loop comprises SEQ ID NO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(d) the sequence of the BC loop comprises SEQ ID NO: 85, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(e) the sequence of the BC loop comprises SEQ ID NO: 86, the sequence of the DE loop comprises SEQ ID NO: 96, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(f) the sequence of the BC loop comprises SEQ ID NO: 87, the sequence of the DE loop comprises SEQ ID NO: 97, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(g) the sequence of the BC loop comprises SEQ ID NO: 88, the sequence of the DE loop comprises SEQ ID NO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(h) the sequence of the BC loop comprises SEQ ID NO: 89, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(i) the sequence of the BC loop comprises SEQ ID NO: 90, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(j) the sequence of the BC loop comprises SEQ ID NO: 91, the sequence of the DE loop comprises SEQ ID NO: 95, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139;
(k) the sequence of the BC loop comprises SEQ ID NO: 92, the sequence of the DE loop comprises SEQ ID NO: 98, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139; or,
(l) the sequence of the BC loop comprises SEQ ID NO: 93, the sequence of the DE loop comprises SEQ ID NO: 94, and the sequence of the FG loop comprises SEQ ID NO: 9 or 139.
27. The method of claim 1 , wherein the CD40L-specific monomer subunit comprises a sequence selected from the group consisting of SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 146.
28. The method of claim 8 , wherein the two CD40L-specific monomer subunits are connected in tandem by a peptide linker.
29. The method of claim 28 , wherein the peptide linker comprises a (GmX)n sequence wherein:
(a) X is Serine (S), Alanine (A), Glycine (G), Leu (L), Isoleucine (I), or Valine (V);
(b) m and n are integers;
(c) m is 1, 2, 3 or 4; and
(d) n is 1, 2, 3, 4, 5, 6, or 7.
30. The method of claim 29 , wherein the peptide linker is selected from the group consisting of: SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 142, and SEQ ID NO: 143.
31. The method of claim 1 , wherein the CD40L-specific monomer subunit comprises seven beta strands designated A, B, C, D, E, F, and G, wherein the A beta strand consists of SEQ ID NO:11, the B beta strand consists of SEQ ID NO:12, the C beta strand consists of SEQ ID NO:13 or SEQ ID NO:14, the D beta strand consists of SEQ ID NO:15, the E beta strand consists of SEQ ID NO:16, the F beta strand consists of SEQ ID NO:17, and the G beta strand consists of SEQ ID NO:18, and six loop regions designated AB, BC, CD, DE, EF, and FG, wherein the AB loop consists of SEQ ID NO: 4, the BC loop consists of SEQ ID NO: 86, the CD loop consists of SEQ ID NO: 6, the DE loop consists of SEQ ID NO: 96, the EF loop consists of SEQ ID NO: 8, and the FG loop consists of SEQ ID NO: 139.
32. The method of claim 1 , wherein the at least one CD40L-specific monomer subunit comprises SEQ ID NO: 146.
33. The method of claim 1 , wherein the Tn3 scaffold comprises SEQ ID NO: 145.
34. A method, comprising administering an effective amount of a composition comprising a Tn3 scaffold to a subject with a disorder comprising inflammation, wherein the Tn3 scaffold comprises at least one CD40L-specific monomer subunit, wherein the monomer subunit comprises seven beta strands designated A, B, C, D, E, F, and G, and six loop regions designated AB, BC, CD, DE, EF, and FG, wherein the AB loop comprises SEQ ID NO: 4, the BC loop comprises a sequence selected from the group consisting of SEQ ID NOS: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, and 168, the CD loop comprises SEQ ID NO: 6, the DE loop comprises a sequence selected from the group consisting of SEQ ID NOs: 94, 95, 96, 97, 98, and 169, the EF loop comprises SEQ ID NO: 8, and the FG loop comprises a sequence selected from the group consisting of SEQ ID NOS: 9, 99, 139, and 170.
35. The method of claim 34 , wherein the disorder is cancer.
36. The method of claim 34 , wherein the disorder is selected from the group consisting of: asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergy, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, inflammatory osteolysis, viral infection, and bacterial infection.
37. The method of claim 34 , wherein the monomer subunit comprises seven beta strands designated A, B, C, D, E, F, and G, wherein the A beta strand consists of SEQ ID NO:11, the B beta strand consists of SEQ ID NO:12, the C beta strand consists of SEQ ID NO:13 or SEQ ID NO:14, the D beta strand consists of SEQ ID NO:15, the E beta strand consists of SEQ ID NO:16, the F beta strand consists of SEQ ID NO:17, and the G beta strand consists of SEQ ID NO:18, and six loop regions designated AB, BC, CD, DE, EF, and FG, wherein the AB loop consists of SEQ ID NO: 4, the BC loop consists of SEQ ID NO: 86, the CD loop consists of SEQ ID NO: 6, the DE loop consists of SEQ ID NO: 96, the EF loop consists of SEQ ID NO: 8, and the FG loop consists of SEQ ID NO: 139.
38. The method of claim 34 , wherein the CD40L-specific monomer subunit comprises SEQ ID NO: 146.
39. The method of claim 34 , wherein the Tn3 scaffold comprises SEQ ID NO: 145.