US2013089513A1PendingUtilityA1

Method of improving efficacy of biological response-modifying proteins and the exemplary muteins

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Assignee: CHUNG YONG-HOONPriority: Jul 26, 2003Filed: Apr 2, 2012Published: Apr 11, 2013
Est. expiryJul 26, 2023(expired)· nominal 20-yr term from priority
Inventors:Yong-Hoon ChungHak-Sup LeeKi-Wan YiYoun-Hwa HeoJae Youn Kim
A61P 7/06C07K 14/535C07K 14/715A61K 38/00C07K 14/524C07K 14/52A61P 25/14A61P 7/00
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Claims

Abstract

Disclosed is a protein variant which substitutes valine for phenylalanine residue in a binding domain having a biological response-modifying function by binding to a receptor, ligand or substrate. Also, the present invention discloses a DNA encoding the protein variant, a recombinant expression vector to which the DNA is operably linked, a host cell transformed or transfected with the recombinant expression vector, and a method of preparing the protein variant comprising cultivating the host cell and isolating the protein variant from the resulting culture. Further, the present invention discloses a pharmaceutical composition comprising the protein variant and a pharmaceutically acceptable carrier.

Claims

exact text as granted — not AI-modified
1 - 9 . (canceled) 
     
     
         10 . A protein variant, wherein a valine residue replaces a phenylalanine residue in a binding domain of a protein having a biological response-modifying function when bound to a receptor, ligand, or substrate. 
     
     
         11 . The protein variant according to  claim 10 , wherein the protein is a cytokine. 
     
     
         12 . The protein variant according to  claim 11 , wherein the cytokine is a 4-alpha helix bundle cytokine. 
     
     
         13 . The protein variant according to  claim 12 , wherein the 4-alpha helix bundle cytokine is selected from the group consisting of CNTF, EPO, Flt3L, G-CSF, GM-CSF, GH, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12p35, LPT, LIF, M-CSF, OSM, PL, SCF, TPO, IFN-α2A, IFN-α2B, IFN-β, IFN-γ, IFN-ω, and IFN-τ. 
     
     
         14 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is CNTF, EPO, Flt3L, G-CSF, GM-CSF, GH, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12p35, LPT, LIF, M-CSF, OSM, PL, SCF or TPO and the valine residue replaces a phenylalanine residue at an amino acid residue between positions 110 and 180. 
     
     
         15 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IFN-α2A, IFN-α2B, IFN-β, IFN-γ, IFN-ω or IFN-τ and the valine residue replaces the phenylalanine residue at an amino acid residue between positions 1 and 50. 
     
     
         16 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is CNTF and the valine residue replaces the phenylalanine residue at position 3, 83, 98, 105, 119, 152, or 178 of SEQ ID NO.: 1. 
     
     
         17 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is EPO and the valine residue replaces the phenylalanine residue at position 48, 138, 142, or 148 of SEQ ID NO.: 2. 
     
     
         18 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is Flt3L and the valine residue replaces the phenylalanine residue at position 6, 15, 81, 87, 96, or 124 of SEQ ID NO.: 3. 
     
     
         19 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is G-CSF and the valine residue replaces the phenylalanine residue at position 13, 83, 113, 140, 144, or 160 of SEQ ID NO.: 4. 
     
     
         20 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is GM-CSF and the valine residue replaces the phenylalanine residue at position 47, 103, 106, 113, or 119 of SEQ ID NO.: 5. 
     
     
         21 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is GH and the valine residue replaces the phenylalanine residue at position 1, 10, 25, 31, 44, 54, 92, 97, 139, 146, 166, 176, or 191 of SEQ ID NO.: 6. 
     
     
         22 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IL-2 and the valine residue replaces the phenylalanine residue at position 42, 44, 78, 103, 117, or 124 of SEQ ID NO.: 13. 
     
     
         23 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IL-3 and the valine residue replaces the phenylalanine residue at position 37, 61, 107, 113, or 133 of SEQ ID NO.: 14. 
     
     
         24 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IL-4 and the valine residue replaces the phenylalanine residue at position 33, 45, 55, 73, 82, or 112 of SEQ ID NO.: 15. 
     
     
         25 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IL-5 and the valine residue replaces the phenylalanine residue at position 49, 69, 96, or 103 of SEQ ID NO.: 16. 
     
     
         26 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IL-6 and the valine residue replaces the phenylalanine residue at position 73, 77, 93, 104, 124, 169, or 172 of SEQ ID NO.: 17. 
     
     
         27 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IL-12p35 and the valine residue replaces the phenylalanine residue at position 13, 39, 82, 96, 116, 132, 150, 166, or 180 of SEQ ID NO.: 18. 
     
     
         28 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is LPT and the valine residue replaces the phenylalanine residue at position 41 or 92 of SEQ ID NO.: 19. 
     
     
         29 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is LIF and the valine residue replaces the phenylalanine residue at position 41, 52, 67, 70, 156, or 180 of SEQ ID NO.: 20. 
     
     
         30 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is M-CSF and the valine residue replaces the phenylalanine residue at position 35, 37, 54, 67, 91, 106, 121, 135, 143, 229, 255, 311, 439, 466, or 485 of SEQ ID NO.: 21. 
     
     
         31 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is OSM and the valine residue replaces the phenylalanine residue at position 56, 70, 160, 169, 176, or 184 of SEQ ID NO.: 22. 
     
     
         32 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is PL and the valine residue replaces the phenylalanine residue at position 10, 31, 44, 52, 54, 92, 97, 146, 166, 176, or 191 of SEQ ID NO.: 23. 
     
     
         33 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is SCF and the valine residue replaces the phenylalanine residue at position 63, 102, 110, 115, 116, 119, 126, 129, 158, 199, 205, 207, or 245 of SEQ ID NO.: 24. 
     
     
         34 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is TPO and the valine residue replaces the phenylalanine residue at position 141, 186, 204, 240, or 286 of SEQ ID NO.: 25. 
     
     
         35 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IFN-α2A and the valine residue replaces the phenylalanine residue at position 27, 36, 38, 43, 47, 64, 67, 84, 123, or 151 of SEQ ID NO.: 7. 
     
     
         36 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IFN-α2B and the valine residue replaces the phenylalanine residue at position 27, 36, 38, 43, 47, 64, 67, 84, 123, or 151 of SEQ ID NO.: 8. 
     
     
         37 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IFN-β and the valine residue replaces the phenylalanine residue at position 8, 38, 50, 67, 70, 111, or 154 of SEQ ID NO.: 9. 
     
     
         38 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IFN-γ and the valine replaces the phenylalanine residue at position 18, 32, 55, 57, 60, 63, 84, 85, 95, or 139 of SEQ ED NO.: 10. 
     
     
         39 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IFN-ω and the valine replaces the phenylalanine residue at position 27, 36, 38, 65, 68, 124, or 153 of SEQ ID NO.: 11. 
     
     
         40 . The protein variant according to  claim 13 , wherein the 4-alpha helix bundle cytokine is IFN-τ and the valine replaces the phenylalanine residue at position 8, 39, 68, 71, 88, 127, 156, 157, 159, or 183 of SEQ ID NO.: 12. 
     
     
         41 . A DNA molecule encoding the protein variant according to  claim 10 . 
     
     
         42 . A recombinant expression vector to which the DNA molecule according to  claim 41  is operably linked. 
     
     
         43 . The recombinant expression vector according to  claim 42 , wherein the recombinant expression vector has an accession number KCCM-10500, KCCM-10501 or KCCM-10571. 
     
     
         44 . A host cell transformed or transfected with the recombinant expression vector according to  claim 42  or  43 . 
     
     
         45 . A method of preparing a protein variant, comprising cultivating the host cell according to  claim 44  and isolating the protein variant from a resulting culture. 
     
     
         46 . A pharmaceutical composition comprising the protein variant according to  claim 10  and a pharmaceutically acceptable carrier.

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