ANTIGENIC POLYPEPTIDES AND METHODS OF USE THEREOF

Information

  • Patent Application
  • 20220257733
  • Publication Number
    20220257733
  • Date Filed
    January 24, 2022
    2 years ago
  • Date Published
    August 18, 2022
    a year ago
Abstract
Provided are novel antigenic polypeptides comprising tumor-associated peptides, and compositions comprising the same. Such antigenic polypeptides and compositions are particularly useful as immunotherapeutics (e.g., cancer vaccines). Also provided are methods of inducing a cellular immune response using the polypeptides and compositions, methods of treating a disease using the polypeptides and compositions, kits comprising the polypeptides and compositions, methods of making the compositions, and antibodies and T cell receptors that specifically bind to the polypeptides.
Description
2. SEQUENCE LISTING

The sequence listing attached herewith, named 404293_AGBW_141US_188624_Sequence_Listing.txt and created on Jul. 24, 2020, is herein incorporated by reference in its entirety.


3. FIELD

The instant disclosure relates to novel antigenic polypeptides and compositions, and uses of such antigenic polypeptides and compositions as immunotherapeutics (e.g., cancer vaccines).


4. BACKGROUND

Immunotherapies are becoming important tools in the treatment of cancer. One immunotherapy approach involves the use of therapeutic cancer vaccines comprising cancer-specific antigenic peptides that actively educate a patient's immune system to target and destroy cancer cells. However, the generation of such therapeutic cancer vaccines is limited by the availability of immunogenic cancer-specific antigenic peptides.


Accordingly, there is a need in the art for improved immunogenic cancer-specific peptides and for creating effective anti-cancer vaccines comprising these peptides.


5. SUMMARY OF INVENTION

The instant disclosure provides novel antigenic polypeptides comprising tumor-associated peptides, and compositions comprising the same. Such antigenic polypeptides and compositions are particularly useful as immunotherapeutics (e.g., cancer vaccines). Also provided are methods of inducing a cellular immune response using the polypeptides and compositions, methods of treating a disease using the polypeptides and compositions, kits comprising the polypeptides and compositions, methods of making the compositions, and antibodies and T cell receptors that specifically bind to the polypeptides.


Accordingly, the instant disclosure provides the following, non-limiting, embodiments:


Embodiment 1. An antigenic polypeptide of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length, comprising an MHC-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171.


Embodiment 2. The antigenic polypeptide of embodiment 1, wherein the amino acid sequence of the MHC-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171.


Embodiment 3. The antigenic polypeptide of embodiment 1, wherein the amino acid sequence of the antigenic polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171.


Embodiment 4. The antigenic polypeptide of embodiment 1 or 2, further comprising an HSP-binding peptide.


Embodiment 5. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein X1 is omitted, N, F, or Q; X2 is W, L, or F; X3 is L or I; X4 is R, L, or K; X5 is L, W, or I; X6 is T, L, F, K, R, or W; and X7 is W, G, K, or F.


Embodiment 6. The antigenic polypeptide of embodiment 5, wherein the HSP-binding peptide comprises the amino acid sequence of:


(a) X1LX2LTX3 (SEQ ID NO: 2), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;


(b) NX1LX2LTX3 (SEQ ID NO: 3), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;


(c) WLX1LTX2 (SEQ ID NO: 4), wherein X1 is R or K; and X2 is W or G;


(d) NWLX1LTX2 (SEQ ID NO: 5), wherein X1 is R or K; and X2 is W or G; or


(e) NWX1X2X3X4X5 (SEQ ID NO: 6), wherein X1 is L or I; X2 is L, R, or K; X3 is L or I; X4 is T, L, F, K, R, or W; and X5 is W or K.


Embodiment 7. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-42, optionally wherein the amino acid sequence of the HSP-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-42.


Embodiment 8. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 7, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 7.


Embodiment 9. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 8, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 8.


Embodiment 10. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 9, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 9.


Embodiment 11. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 10, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 10.


Embodiment 12. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 11, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 11.


Embodiment 13. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 12, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 12.


Embodiment 14. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 13, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 13.


Embodiment 15. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 14, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 14.


Embodiment 16. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 15, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 15.


Embodiment 17. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 16, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 16.


Embodiment 18. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 17, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 17.


Embodiment 19. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 18, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 18.


Embodiment 20. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 19, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 19.


Embodiment 21. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 20, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 20.


Embodiment 22. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 21, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 21.


Embodiment 23. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 22, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 22.


Embodiment 24. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 23, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 23.


Embodiment 25. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 24, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 24.


Embodiment 26. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 25, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 25.


Embodiment 27. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 26, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 26.


Embodiment 28. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 27, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 27.


Embodiment 29. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 28, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 28.


Embodiment 30. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 29, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 29.


Embodiment 31. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 30, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 30.


Embodiment 32. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 31, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 31.


Embodiment 33. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 32, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 32.


Embodiment 34. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 33, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 33.


Embodiment 35. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 34, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 34.


Embodiment 36. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 35, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 35.


Embodiment 37. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 36, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 36.


Embodiment 38. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 37, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 37.


Embodiment 39. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 38, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 38.


Embodiment 40. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 39, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 39.


Embodiment 41. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 40, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 40.


Embodiment 42. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 41, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 41.


Embodiment 43. The antigenic polypeptide of embodiment 4, wherein the HSP-binding peptide comprises the amino acid sequence of SEQ ID NO: 42, optionally wherein the amino acid sequence of the HSP-binding peptide consists of the amino acid sequence of SEQ ID NO: 42.


Embodiment 44. The antigenic polypeptide of any one of the preceding embodiments, wherein the MHC-binding peptide is 8 to 50 amino acids in length, optionally 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.


Embodiment 45. The antigenic polypeptide of any one of embodiments 4-44, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the HSP-binding peptide.


Embodiment 46. The antigenic polypeptide of any one of embodiments 4-44, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the HSP-binding peptide.


Embodiment 47. The antigenic polypeptide of any one of embodiments 4-46, wherein the HSP-binding peptide is linked to the MHC-binding peptide via a chemical linker.


Embodiment 48. The antigenic polypeptide of any one of embodiments 4-46, wherein the HSP-binding peptide is linked to the MHC-binding peptide via a peptide linker.


Embodiment 49. The antigenic polypeptide of embodiment 48, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 43, optionally wherein the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 43.


Embodiment 50. The antigenic polypeptide of embodiment 48, wherein the peptide linker comprises the amino acid sequence of FR, optionally wherein the amino acid sequence of the peptide linker consists of the amino acid sequence of FR.


Embodiment 51. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of:

  • (a) the amino acid sequence of X1X2X3X4X5X6X7FFRK (SEQ ID NO: 68), wherein X1 is omitted, N, F, or Q; X2 is W, L, or F; X3 is L or I; X4 is R, L, or K; X5 is L, W, or I; X6 is T, L, F, K, R, or W; and X7 is W, G, K, or F;
  • (b) the amino acid sequence of X1LX2LTX3FFRK (SEQ ID NO: 69), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;
  • (c) the amino acid sequence of NX1LX2LTX3FFRK (SEQ ID NO: 70), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;
  • (d) the amino acid sequence of WLX1LTX2FFRK (SEQ ID NO: 71), wherein X1 is R or K; and X2 is W or G;
  • (e) the amino acid sequence of NWLX1LTX2FFRK (SEQ ID NO: 72), wherein X1 is R or K; and X2 is W or G;
  • (f) the amino acid sequence of NWX1X2X3X4X5FFRK (SEQ ID NO: 73), wherein X1 is L or I; X2 is L, R, or K; X3 is L or I; X4 is T, L, F, K, R, or W; and X5 is W or K; or
  • (g) an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-97.


    Embodiment 52. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 74.


    Embodiment 53. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 75.


    Embodiment 54. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 76.


    Embodiment 55. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 77.


    Embodiment 56. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 78.


    Embodiment 57. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 79.


    Embodiment 58. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 80.


    Embodiment 59. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 81.


    Embodiment 60. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 82.


    Embodiment 61. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 83.


    Embodiment 62. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 84.


    Embodiment 63. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 85.


    Embodiment 64. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 86.


    Embodiment 65. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 87.


    Embodiment 66. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 88.


    Embodiment 67. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 89.


    Embodiment 68. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 90.


    Embodiment 69. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 91.


    Embodiment 70. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 92.


    Embodiment 71. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 93.


    Embodiment 72. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 94.


    Embodiment 73. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 95.


    Embodiment 74. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 96.


    Embodiment 75. The antigenic polypeptide of embodiment 49 or 50, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of the amino acid sequence set forth in SEQ ID NO: 97.


    Embodiment 76. The isolated polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of:
  • (a) the amino acid sequence of FFRKX1X2X3X4X5X6X7 (SEQ ID NO: 44), wherein X1 is omitted, N, F, or Q; X2 is W, L, or F; X3 is L or I; X4 is R, L, or K; X5 is L, W, or I; X6 is T, L, F, K, R, or W; and X7 is W, G, K, or F;
  • (b) the amino acid sequence of FFRKX1LX2LTX3 (SEQ ID NO: 45), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;
  • (c) the amino acid sequence of FFRKNX1LX2LTX3 (SEQ ID NO: 46), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;
  • (d) the amino acid sequence of FFRKWLX1LTX2 (SEQ ID NO: 47), wherein X1 is R or K; and X2 is W or G;
  • (e) the amino acid sequence of FFRKNWLX1LTX2 (SEQ ID NO: 48), wherein X1 is R or K; and X2 is W or G;
  • (f) the amino acid sequence of FFRKNWX1X2X3X4X5 (SEQ ID NO: 49), wherein X1 is L or I; X2 is L, R, or K; X3 is L or I; X4 is T, L, F, K, R, or W; and X5 is W or K; or
  • (g) an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-67.


    Embodiment 77. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 50.


    Embodiment 78. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 51.


    Embodiment 79. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 52.


    Embodiment 80. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 53.


    Embodiment 81. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 54.


    Embodiment 82. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 55.


    Embodiment 83. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 56.


    Embodiment 84. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 57.


    Embodiment 85. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 58.


    Embodiment 86. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 59.


    Embodiment 87. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 60.


    Embodiment 88. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 61.


    Embodiment 89. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 62.


    Embodiment 90. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 63.


    Embodiment 91. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 64.


    Embodiment 92. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 65.


    Embodiment 93. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 66.


    Embodiment 94. The antigenic polypeptide of embodiment 49 or 50, wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of the amino acid sequence set forth in SEQ ID NO: 67.


    Embodiment 95. The antigenic polypeptide of embodiment 4, wherein the amino acid sequence of the antigenic polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1372-3919, 3997-4148, and 4172-4217.


    Embodiment 96. The antigenic polypeptide of any one of the preceding embodiments, wherein the antigenic polypeptide is 8 to 50 amino acids in length, optionally 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.


    Embodiment 97. The antigenic polypeptide of embodiment 4, wherein the amino acid sequence of the antigenic polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1372-3919, 3997-4148, and 4172-4217.


    Embodiment 98. The antigenic polypeptide of any one of the preceding embodiments, wherein the antigenic polypeptide is chemically synthesized.


    Embodiment 99. The antigenic polypeptide of any one of the preceding embodiments, wherein a phosphorylated amino acid residue of the phosphopeptide is replaced by a non-hydrolyzable mimetic of the phosphorylated amino acid residue.


    Embodiment 100. A composition comprising at least one of the antigenic polypeptides of any one of embodiments 1-99.


    Embodiment 101. A composition comprising a complex of the antigenic polypeptide of any one of embodiments 1-99 and a purified stress protein.


    Embodiment 102. The composition of embodiment 101, wherein the stress protein is selected from the group consisting of Hsc70, Hsp70, Hsp90, Hsp110, Grp170, Gp96, Calreticulin, and a mutant or fusion protein thereof.


    Embodiment 103. The composition of embodiment 102, wherein the stress protein is an Hsc70, optionally a human Hsc70.


    Embodiment 104. The composition of embodiment 103, wherein the Hsc70 comprises the amino acid sequence of SEQ ID NO: 3920.


    Embodiment 105. The composition of embodiment 103, wherein the amino acid sequence of the Hsc70 consists of the amino acid sequence of SEQ ID NO: 3920.


    Embodiment 106. The composition of any one of embodiments 101-105, wherein the stress protein is a recombinant protein.


    Embodiment 107. The composition any one of embodiments 100-106, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 different antigenic polypeptides.


    Embodiment 108. The composition of embodiment 107, wherein each of the different polypeptides comprise the same HSP-binding peptide and a different MHC-binding peptide.


    Embodiment 109. The composition of any one of embodiments 100-108, wherein the total amount of the polypeptide(s) in the composition is about 0.1 to 20 nmol, optionally about 3, 4, 5, or 6 nmol.


    Embodiment 110. The composition of any one of embodiments 101-109, wherein the amount of the stress protein in the composition is about 10 μg to 600 μg, optionally about 120 μg, 240 μg, or 480 μg.


    Embodiment 111. The composition of any one of embodiments 101-110, wherein the molar ratio of the antigenic polypeptide(s) to the stress protein is about 0.5:1 to about 5:1, optionally about 1:1, 1.25:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1.


    Embodiment 112. The composition of any one of embodiments 100-111, wherein the composition further comprises an adjuvant.


    Embodiment 113. The composition of embodiment 112, wherein the adjuvant comprises a saponin or an immunostimulatory nucleic acid.


    Embodiment 114. The composition of embodiment 113, wherein the adjuvant comprises QS-21.


    Embodiment 115. The composition of embodiment 114, wherein the amount of the QS-21 in the composition is about 10 μg to about 200 μg, optionally about 25 μg, 50 μg, 75 μg, 100 μg, 125 μg, 150 μg, 175 μg, or 200 μg.


    Embodiment 116. The composition of any one of embodiments 112-115, wherein the adjuvant comprises a TLR agonist, optionally a TLR4 agonist, TLR5 agonist, TLR7 agonist, TLR8 agonist, and/or TLR9 agonist.


    Embodiment 117. The composition of any one of embodiments 100-116, further comprising a pharmaceutically acceptable carrier or excipient.


    Embodiment 118. The composition of embodiment 117, wherein the composition is in a unit dosage form.


    Embodiment 119. A method of inducing a cellular immune response to an antigenic polypeptide in a subject, the method comprising administering to the subject an effective amount of the antigenic polypeptide of any one of embodiments 1-99 or the composition of any one of embodiments 100-118.


    Embodiment 120. The method of embodiment 119, wherein the subject has cancer, optionally Acute Myeloid Leukemia (AML) or colorectal cancer.


    Embodiment 121. A method of treating a disease in a subject, the method comprising administering to the subject an effective amount of the antigenic polypeptide of any one of embodiments 1-99 or the composition of any one of embodiments 100-118.


    Embodiment 122. The method of embodiment 121, wherein the disease is cancer, optionally AML or colorectal cancer.


    Embodiment 123. The method of any one of embodiments 119-122, wherein the composition is administered to the subject weekly for four weeks.


    Embodiment 124. The method of embodiment 123, wherein at least two further doses of the composition are administered biweekly to the subject after the four weekly doses.


    Embodiment 125. The method of embodiment 123 or 124, wherein at least one booster dose of the composition is administered three months after the final weekly or biweekly dose.


    Embodiment 126. The method of embodiment 125, wherein the composition is further administered every three months for at least 1 year.


    Embodiment 127. The method of any one of embodiments 119-126, further comprising administering to the subject lenalidomide, dexamethasone, interleukin-2, recombinant interferon alfa-2b, or PEG-interferon alfa-2b.


    Embodiment 128. The method of any one of embodiments 119-127, further comprising administering to the subject an indoleamine dioxygenase-1 (IDO-1) inhibitor.


    Embodiment 129. The method of embodiment 128, wherein the IDO-1 inhibitor is 4-amino-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide.


    Embodiment 130. The method of any one of embodiments 119-129, further comprising administering to the subject an immune checkpoint antibody.


    Embodiment 131. The method of embodiment 130, wherein the immune checkpoint antibody is selected from the group consisting of an agonistic anti-GITR antibody, an agonistic anti-OX40 antibody, an antagonistic anti-PD-1 antibody, an antagonistic anti-CTLA-4 antibody, an antagonistic anti-TIM-3 antibody, an antagonistic anti-LAG-3 antibody, an antagonistic anti-TIGIT antibody, an agonistic anti-CD96 antibody, an antagonistic anti-VISTA antibody, an antagonistic anti-CD73 antibody, an agonistic anti-CD137 antibody, an antagonist anti-CEACAM1 antibody, an agonist anti-ICOS antibody, and an antigen-binding fragment thereof.


    Embodiment 132. A kit comprising a first container containing the polypeptide of any one of embodiments 1-99, or the composition of any one of embodiments 100-118 and a second container containing a purified stress protein capable of binding to the polypeptide.


    Embodiment 133. The kit of embodiment 132, wherein the total amount of the polypeptide(s) in the first container is about 0.1 to 20 nmol, optionally about 3, 4, 5, or 6 nmol.


    Embodiment 134. The kit of embodiment 132 or 133, wherein the stress protein is selected from the group consisting of Hsc70, Hsp70, Hsp90, Hsp110, Grp170, Gp96, Calreticulin, and a mutant or fusion protein thereof.


    Embodiment 135. The kit of embodiment 134, wherein the stress protein is an Hsc70, optionally human a Hsc70.


    Embodiment 136. The kit of embodiment 135, wherein the Hsc70 comprises the amino acid sequence of SEQ ID NO: 3920.


    Embodiment 137. The kit of embodiment 135, wherein the amino acid sequence of the Hsc70 consists of the amino acid sequence of SEQ ID NO: 3920.


    Embodiment 138. The kit of any one of embodiments 132-137, wherein the stress protein is a recombinant protein.


    Embodiment 139. The kit of any one of embodiments 132-138, wherein the amount of the stress protein in the second container is about 10 μg to 600 μg, optionally about 120 μg, 240 μg, or 480 μg.


    Embodiment 140. The kit of any one of embodiments 132-139, wherein the molar ratio of the polypeptide to the stress protein is about 0.5:1 to 5:1, optionally about 1:1, 1.25:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1.


    Embodiment 141. The kit of any one of embodiments 132-140, further comprising a third container containing an adjuvant.


    Embodiment 142. The kit of embodiment 141, wherein the adjuvant comprises a saponin or an immunostimulatory nucleic acid.


    Embodiment 143. The kit of embodiment 142, wherein the adjuvant comprises QS-21.


    Embodiment 144. The kit of embodiment 143, wherein the amount of the QS-21 in the third container is about 10 μg to about 200 μg, optionally about 25 μg, 50 μg, 75 μg, 100 μg, 125 μg, 150 μg, 175 μg, or 200 μg.


    Embodiment 145. The kit of any one of embodiments 141-144, wherein the adjuvant comprises a TLR agonist, optionally a TLR4 agonist, TLR5 agonist, TLR7 agonist, TLR8 agonist, and/or TLR9 agonist.


    Embodiment 146. A method of making a vaccine, the method comprising mixing one or more of the polypeptides of any one of embodiments 1-99, or the composition of any one of embodiments 100-118, with a purified stress protein under suitable conditions such that the purified stress protein binds to at least one of the polypeptides.


    Embodiment 147. The method of embodiment 146, wherein the stress protein is selected from the group consisting of Hsc70, Hsp70, Hsp90, Hsp110, Grp170, Gp96, Calreticulin, and a mutant or fusion protein thereof.


    Embodiment 148. The method of embodiment 147, wherein the stress protein is an Hsc70, optionally a human Hsc70.


    Embodiment 149. The method of embodiment 148, wherein the Hsc70 comprises the amino acid sequence of SEQ ID NO: 3920.


    Embodiment 150. The method of embodiment 148, wherein the amino acid sequence of the Hsc70 consists of the amino acid sequence of SEQ ID NO: 3920.


    Embodiment 151. The method of any one of embodiments 146-150, wherein the stress protein is a recombinant protein.


    Embodiment 152. The method of any one of embodiments 146-151, wherein the molar ratio of the polypeptide to the stress protein is about 0.5:1 to 5:1, optionally about 1:1, 1.25:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1.


    Embodiment 153. The method of any one of embodiments 146-152, wherein the suitable conditions comprise a temperature of about 37° C.


    Embodiment 154. An isolated antibody that: (i) specifically binds to an MHC-binding peptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, optionally wherein the antibody does not specifically bind to an unphosphorylated variant of the MHC-binding peptide; and/or (ii) specifically binds to a complex of an MHC molecule and an MHC-binding peptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, optionally wherein the antibody does not specifically bind to a complex of an MHC molecule and an unphosphorylated variant of the MHC-binding peptide.


    Embodiment 155. The antibody of embodiment 154, which is a chimeric antigen receptor.


    Embodiment 156. An isolated T cell receptor (TCR) that specifically binds to a complex of an MHC molecule and an MHC-binding peptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, optionally wherein the TCR does not specifically bind to a complex of the MHC molecule and an unphosphorylated variant of the MHC-binding peptide.


    Embodiment 157. The TCR of embodiment 156, which is a soluble TCR.


    Embodiment 158. The TCR of embodiment 156 or 157, further comprising a CD3 binding moiety.


    Embodiment 159. An isolated polynucleotide encoding a VH and/or VL of the antibody of embodiment 154 or 155.


    Embodiment 160. An isolated polynucleotide encoding a variable region, optionally a Va and/or VO, of the TCR of any one of embodiments 156-158.


    Embodiment 161. The isolated polynucleotide of embodiment 159 or 160, which is an mRNA.


    Embodiment 162. A vector comprising the polynucleotide of embodiment 159 or 160.


    Embodiment 163. An engineered cell comprising the antibody of embodiment 154 or 155, or the TCR of any one of embodiments 156-158.


    Embodiment 164. An engineered cell comprising the polynucleotide of any one of embodiments 159-161 or the vector of embodiment 162.


    Embodiment 165. The engineered cell of embodiment 163 or 164, wherein the cell is a human lymphocyte.


    Embodiment 166. The engineered cell of any one of embodiments 163-165, wherein the cell is selected from the group consisting of a T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, a mucosal-associated invariant T (MAiT) cell, and a natural killer (NK) cell.







6. DETAILED DESCRIPTION

The instant disclosure provides novel antigenic polypeptides comprising tumor-associated peptides, and compositions comprising the same. Such antigenic polypeptides and compositions are particularly useful as immunotherapeutics (e.g., cancer vaccines). Also provided are methods of inducing a cellular immune response using the polypeptides and compositions, methods of treating a disease using the polypeptides and compositions, kits comprising the polypeptides and compositions, methods of making the compositions, and antibodies and T cell receptors that specifically bind to the polypeptides.


6.1 Definitions

Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting.


As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of 5% to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., up to 5% to 10% below) the recited value or range remain within the intended meaning of the recited value or range.


As used herein, the term “antigenic polypeptide” refers to a polymer comprising one or more MHC-binding peptides. An antigenic polypeptide can comprise one or more non-amino-acid-residue structures. In certain embodiments, an antigenic polypeptide comprises a chemical linker, e.g., a chemical linker linking two peptide portions of the antigenic polypeptide.


As used herein, the terms “major histocompatibility complex” and “MHC” are used interchangeably and refer to an MHC class I molecule and/or an MHC class II molecule.


As used herein, the terms “human leukocyte antigen” and “HLA” are used interchangeably and refer to major histocompatibility complex (MHC) in humans. An HLA molecule may be a class I MHC molecule (e.g., HLA-A, HLA-B, HLA-C) or a class II MHC molecule (e.g., HLA-DP, HLA-DQ, HLA-DR).


As used herein, the term “MHC-binding peptide” refers to a peptide that binds to or is predicted to bind to an MHC molecule, e.g., such that the peptide is capable of being presented by the MHC molecule to a T-cell.


As used herein, the term “HSP-binding peptide” refers to a peptide that non-covalently binds to a heat shock protein (HSP).


As used herein, the term “peptide linker” refers to a peptide bond or a peptide sequence that links a C-terminal amino acid residue of a first peptide to an N-terminal amino acid residue of a second peptide.


As used herein, the term “chemical linker” refers to any chemical bond or moiety that is capable of linking two molecules (e.g., two peptides), wherein the bond or moiety is not a peptide linker.


As used herein, the term “isolated” with respect to a polypeptide, polynucleotide, antibody, or T cell receptor, refers to polypeptide, polynucleotide, antibody, or T cell receptor, that is separated from at least one impurity, e.g., an impurity found together with the molecule in nature, or present after the expression (e.g., recombinant expression) or synthesis (e.g., chemical synthesis) of the molecule.


As used herein, the terms “antibody” and “antibodies” include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and/or VL regions. Examples of antibodies include, without limitation, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab′)2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the above, and conjugates or fusion proteins comprising any of the above (e.g., a chimeric antigen receptor). In certain embodiments, antibodies described herein refer to polyclonal antibody populations. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, antibodies described herein are IgG antibodies, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody. In certain embodiments, the antibody is chimeric antigen receptor.


As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).


As used herein, the terms “VH region” and “VL region” refer, respectively, to single antibody heavy and light chain variable regions, comprising FR (Framework Regions) 1, 2, 3 and 4 and CDR (Complementarity Determining Regions) 1, 2 and 3 (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is herein incorporated by reference in its entirety).


As used herein, the term “chimeric antigen receptor” refers to a fusion protein comprising one or more antibody variable regions linked to heterologous transmembrane and cytoplasmic regions (e.g., cytoplasmic regions from a T cell costimulatory receptor, such as CD28 or 41BB).


As used herein, the terms “T cell receptor” and “TCR” are used interchangeably and refer to molecules comprising CDRs or variable regions from αβ or γδ T cell receptors. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing variable regions of TCRs attached by a flexible linker, TCR chains linked by an engineered disulfide bond, single TCR variable domains, single peptide-MHC-specific TCRs, multi-specific TCRs (including bispecific TCRs), TCR fusions, TCRs comprising co-stimulatory regions, human TCRs, humanized TCRs, chimeric TCRs, recombinantly produced TCRs, and synthetic TCRs. In certain embodiments, the TCR is a full-length TCR comprising a full-length α chain and a full-length β chain. In certain embodiments, the TCR is a soluble TCR lacking transmembrane and/or cytoplasmic region(s). In certain embodiments, the TCR is a single-chain TCR (scTCR) comprising Vα and Vβ linked by a peptide linker, such as a scTCR having a structure as described in PCT Publication No.: WO 2003/020763, WO 2004/033685, or WO 2011/044186, each of which is incorporated by reference herein in its entirety. In certain embodiments, the TCR comprises a transmembrane region. In certain embodiment, the TCR comprises a co-stimulatory signaling region.


As used herein, the term “full-length TCR” refers to a TCR comprising a dimer of a first and a second polypeptide chain, each of which comprises a TCR variable region and a TCR constant region comprising a TCR transmembrane region and a TCR cytoplasmic region. In certain embodiments, the full-length TCR comprises one or two unmodified TCR chains, e.g., unmodified α, β, γ, or δ TCR chains. In certain embodiments, the full-length TCR comprises one or two altered TCR chains, such as chimeric TCR chains and/or TCR chains comprising one or more amino acid substitutions, insertions, or deletions relative to an unmodified TCR chain. In certain embodiments, the full-length TCR comprises a mature, full-length TCR α chain and a mature, full-length TCR β chain. In certain embodiments, the full-length TCR comprises a mature, full-length TCR γ chain and a mature, full-length TCR δ chain.


As used herein, the term “TCR variable region” refers to the portion of a mature TCR polypeptide chain (e.g., a TCR α chain or β chain) which is not encoded by the TRAC gene for TCR α chains, either the TRBC1 or TRBC2 genes for TCR β chains, the TRDC gene for TCR δ chains, or either the TRGC1 or TRGC2 gene for TCR γ chains. In some embodiments, the TCR variable region of a TCR α chain encompasses all amino acids of a mature TCR α chain polypeptide which are encoded by a TRAV and/or TRAJ gene, and the TCR variable region of a TCR β chain encompasses all amino acids of a mature TCR β chain polypeptide which are encoded by a TRBV, TRBD, and/or TRBJ gene (see, e.g., T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is incorporated by reference herein in its entirety). TCR variable regions generally comprise framework regions (FR) 1, 2, 3 and 4 and complementarity determining regions (CDR) 1, 2 and 3.


As used herein, the terms “α chain variable region” and “Vα” are used interchangeably and refer to the variable region of a TCR α chain.


As used herein, the terms “β chain variable region” and “Vβ” are used interchangeably and refer to the variable region of a TCR β chain.


As used herein, the term “specifically binds to” refers to the ability of an antibody or TCR to preferentially bind to a particular antigen (e.g., a specific MHC-binding polypeptide, or MHC-binding polypeptide/MHC complex) as such binding is understood by one skilled in the art. For example, an antibody or TCR that specifically binds to an antigen can bind to other antigens, generally with lower affinity as determined by, e.g., BIAcore®, or other immunoassays known in the art (see, e.g., Savage et al., Immunity. 1999, 10(4):485-92, which is incorporated by reference herein in its entirety). In a specific embodiment, an antibody or TCR that specifically binds to an antigen binds to the antigen with an association constant (Ka) that is at least 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, or 10,000-fold greater than the Ka when the antibody or TCR binds to another antigen.


As used herein, the terms “treat,” “treating,” and “treatment” refer to methods that generally involve administration of an agent (e.g., a polypeptide disclosed herein) to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder, or in order to prolong the survival of the subject beyond that expected in the absence of such treatment.


As used herein, the term “effective amount” in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect.


As used herein, the term “subject” includes any human or non-human animal.


6.2 Antigenic Polypeptides

In one aspect, the instant disclosure provides an antigenic polypeptide comprising a tumor-associated MHC-binding peptide. Exemplary MHC-binding peptides for use in the antigenic polypeptides disclosed herein are set forth in Table 1 herein.









TABLE 1







Amino acid sequences


of exemplary MHC-binding


peptides








SEQ



ID



NO
Amino Acid Sequence











98
AELGRLsPRAY





99
AESImsFHI





100
AESIMsFHI





101
AEsLKSLSSEL





102
AEtPDIKLF





103
AGFsFVNPK





104
AHDPSGmFRSQsF





105
ALDSGAsLLHL





106
ALmGsPQLVAA





107
ALPPGSYAsL





108
ALPTPALsPSLM





109
ALSsSFLVL





110
ALSSsFLVL





111
ALStPVVEK





112
ALVDGyFRL





113
ALwsPGLAK





114
AmLGSKsPDPYRL





115
APAsPFRQL





116
APAsPLRPL





117
APAsPNHAGVL





118
APFHLtPTLY





119
APKsPSSEWL





120
APRtPPGVTF





121
APsSPDVKL





122
APSsPDVKL





123
APTsPLGHL





124
APVsPRPGL





125
ARFsGFYSm





126
ARFsGFYSM





127
ARFsPKVSL





128
ARGIsPIVF





129
ARYsGSYNDY





130
ASFKAELsY





131
ASFtPTSILK





132
ASFtPTSILKR





133
ASLsPSVSK





134
ATIsPPLQPK





135
AVILPPLsPYFK





136
AVLEyLKI





137
AVNQFsPSLAR





138
AVRNFsPTDYY





139
AVRNFSPtDYY





140
AWRRLsRDSGGY





141
AYGGLtSPGLSY





142
AYGGLTsPGLSY





143
AYSsYVHQY





144
CtFGSRQI





145
DFAsPFHER





146
DFHsPIVLGR





147
DIAsPTFRRL





148
DIIRQPsEEEIIK





149
DIKsVFEAF





150
DILsPRLIR





151
DIRRFsLTTLR





152
DIsPPIFRR





153
DLtLKKEKF





154
DMLGLtKPAMPM





155
DNFsPDLRVLR





156
DPFGRPTsF





157
DPLIRWDsY





158
DPSLDLHsL





159
DSDPmLsPRFY





160
DSDPMLsPRFY





161
DSDPmLsPRFYAY





162
DSDPMLsPRFYAY





163
DsGEGDFLAEGGGVR





164
DSKsPLGFY





165
DTIsLASERY





166
DTIsPTLGF





167
DTQSGsLLFIGR





168
DTsSLPTVIMR





169
DTSsLPTVImR





170
DTSsLPTVIMR





171
DTTsLRTLRI





172
DVAsPDGLGRL





173
DVAsPTLR





174
DVAsPTLRR





175
DVAsPTLRRL





176
DVIDsQELSKV





177
DVYSGtPTKV





178
DYSPYFKtI





179
EAsSPVPYL





180
EASsPVPYL





181
EEAPQtPVAF





182
EEDtYEKVF





183
EEFsPRQAQmF





184
EEFsPRQAQMF





185
EEIsPTKFPGL





186
EEIsPTKFPGLY





187
EELsPLALGRF





188
EELsPSTVLY





189
EELSPsTVLY





190
EELSPtAKF





191
EGPEtGYSL





192
EHERSIsPLLF





193
EIVNFsPIAR





194
ERLKIRGsL





195
ERVDSLVsL





196
ESFSDyPPLGRFA





197
ESLsPIGDmKV





198
ESLsPIGDMKV





199
ESVYKASLsL





200
ETRRPsYLEW





201
EVIRKGsITEY





202
EVIsQHLVSY





203
EVIsVLQKY





204
EVLERKIsM





205
FAFPGStNSL





206
FAFPGSTNsL





207
FASPtSPPVL





208
FASPTsPPVL





209
FATIKSAsL





210
FATIRTAsL





211
FAVsPIPGRGGVL





212
FAwsPLAGEKF





213
FAWsPLAGEKF





214
FAYsPGGAHGmL





215
FFFtARTSF





216
FGGQRLtL





217
FHGISTVsL





218
FHVtPLKL





219
FIVsPVPESRL





220
FKVsPLTFGR





221
FLDsAYFRL





222
FLDsGTIRGV





223
FLFsPPEVTGR





224
FLKPsTSGDSL





225
FLKPSTsGDSL





226
FLKPSTSGDsL





227
FLNEKARLsY





228
FLsRSIPSL





229
FPDNsDVSSIGRL





230
FPDNSDVSsIGRL





231
FPLMRSKsL





232
FPLsPTKLSQY





233
FPSMPsPRL





234
FQYSKSPsL





235
FRFsPMGVDHM





236
FRPPPLtPEDVGF





237
FRRPDIQYPDAtDE





238
FRRsDDMFTF





239
FRYSGKtEY





240
FSFKKsFKL





241
FSFsPGAGAFR





242
FSLRYsPGmDAY





243
FSLRYsPGMDAY





244
FSRPSMsPTPLDR





245
FSVDsPRIY





246
FTIFRTIsV





247
FtPPVVKR





248
FVLsPIKEPA





249
FVRsPGTGAF





250
FVtTPTAEL





251
FVTtPTAEL





252
FVTTPtAEL





253
FYYsPSGKKF





254
GALsRYLFR





255
GEDPLsPRAL





256
GELEsIGELF





257
GEmsPQRFF





258
GEMsPQRFF





259
GEmsPQRFFF





260
GENKsPLLL





261
GEPRAPtPPSGTEVTL





262
GEPsPPHDIL





263
GEtSPRTKIW





264
GETsPRTKITW





265
GEwsASLPHRF





266
GEwSAsLPHRF





267
GEWsASLPHRF





268
GEYsPGTALP





269
GGLTsPGLSY





270
GGSISVQVNSIKFDsE





271
GHGsPFPSL





272
GIFPGtPLKK





273
GIISsPLTGK





274
GIISSPLtGK





275
GImsPLAKK





276
GLFsPIRSSAF





277
GLLsLSALGSQAHL





278
GLPGGGsPTTFL





279
GLSsLSIHL





280
GLTsPGLSYSL





281
GLtVSIPGL





282
GMAILsLLLK





283
GPGHHHKPGLGEGtP





284
GPLSRVKsL





285
GPLVRQIsL





286
GPRAPSPtKPL





287
GPRsASLL





288
GPRSFtPLSI





289
GPRsPKAWL





290
GPRtPTQPLL





291
GRNsLSSLPTY





292
GRQSPsFKL





293
GSFAsPGRLF





294
GsFRGFPAL





295
GSKsPDPYRL





296
GSRsLYNLR





297
GTFPKALsI





298
GtPLSQAIIHQY





299
GTVtPPPRLVK





300
GTYVPSsPTRLAY





301
GVIKsPSWQR





302
GVIsPQELLK





303
GVIsPQELLKK





304
GVLsPDTISSK





305
GVmtPLIKR





306
GVMtPLIKR





307
HEFsSPSHLL





308
HEFSsPSHLL





309
HELsDITEL





310
HERSIsPLL





311
HFDsPPHLL





312
HHHKPGLGEGtP





313
HHPGLGEGtP





314
HKIsDYFEY





315
HLLEtTPKSE





316
HLLETtPKSE





317
HLLSPtKGI





318
HLNsLDVQL





319
HLPsPPLTQEV





320
HLSsFTMKL





321
HPIsPYEHL





322
HPIsPYEHLL





323
HPIsSEELL





324
HPISsEELL





325
HPIsSEELLSLKY





326
HPISsEELLSLKY





327
HPRPVPDsPVSVTRL





328
HPRsPNVLSVAL





329
HPsLSAPAL





330
HPSLsAPAL





331
HPTLQAPsL





332
HPYRNsDPVI





333
HQFsLKENw





334
HQGKFLQtF





335
HRAsKVLFL





336
HRDsFSRmSL





337
HRDsFSRMSL





338
HRNsmKVFL





339
HRVsVILKL





340
HSDKRRPPsAELY





341
HSLsLDDIRLY





342
HSVsPDPVL





343
HTIsPLDLA





344
HTIsPLDLAK





345
HTIsPLDLAKL





346
HTIsPSFQL





347
HTISPsFQL





348
HVSLITPtKR





349
HYFsPFRPY





350
HYsSRLGSAIF





351
HYSsRLGSAIF





352
HYSSRLGsAIF





353
IAATKsLSV





354
IEIERILsV





355
IFDLQKTsL





356
IIQsPSSTGLLK





357
ILGPPPPsFHL





358
ILLtDLII





359
IMKNLQAHyE





360
IPHQRSsL





361
IPKsKFLAL





362
IPMtPTSSF





363
IPMTPtSSF





364
IPRPLsLIG





365
IPRsFRHLSF





366
IPsmSHVHL





367
IPsMSHVHL





368
IPsPLQPEm





369
IPsPLQPEM





370
IPVSKPLsL





371
IPVsRDWEL





372
IRFGRKPsL





373
IRPsVLGPL





374
IRRsYFEVF





375
IRYSGHsL





376
ISKKLsFLSW





377
ISLDKLVsI





378
IsSLTTLSI





379
ISsLTTLSI





380
ISsSmHSLY





381
ISsSMHSLY





382
ISSsmHSLY





383
ITItPPEKY





384
ITLLsPKHKY





385
ItPPSSEKLVSVm





386
ItPPSSEKLVSVM





387
ITTsPITVR





388
ITTsPITVRK





389
ITYsPKLER





390
IVLPLsLQR





391
IVsSLRLAY





392
IVSsLRLAY





393
IYDsVKVYF





394
IYRSQsPHYF





395
KAFsESGSNLHAL





396
KAFsPVRSVR





397
KAFsPVRSVRK





398
KAItPPQQPY





399
KASsPGHPAF





400
KAVsFHLVH





401
KAVsLFL





402
KAYtPVVVTQW





403
KEDsFLQRY





404
KEmSPtRQL





405
KEsEVFYEL





406
KEsTLHLVL





407
KEStLHLVL





408
KFLsPAQYLY





409
KFRDLsPPRY





410
KFsLRAAEF





411
KGFsGTFQL





412
KIFERATsF





413
KIFsKQQGKAFQR





414
KIIsIFSG





415
KIIsIFSGTEK





416
KIKsLEEIYL





417
KINsLAHLR





418
KISsFTSLK





419
KISSFtSLK





420
KISSFTsLK





421
KISsLEIKL





422
KKLsLLNGGL





423
KLEGPDVsL





424
KLFHGsLEEL





425
KLFPGsPAIY





426
KLHsLIGLGI





427
KLIDIVSsQKV





428
KLKsFTYEY





429
KLLDFGsLSNL





430
KLLEGEESRIsL





431
KLLsPILARY





432
KLLsTALHV





433
KLLsYIQRL





434
KLMsDVEDVSL





435
KLMsLGDIRL





436
KLmsPKADVKL





437
KLMsPVLKQHL





438
KLQEFsKEE





439
KLRIQtDGDKY





440
KLSsGLLPKL





441
KLwtLVSEQTRV





442
KLWtLVSEQTRV





443
KLYRPGsVAY





444
KLYsISSQV





445
KLYsPTSKAL





446
KLYSPtSKAL





447
KLYTyIQSR





448
KLYTyIQSRF





449
KmDsFLDMQL





450
KMDsFLDmQL





451
KmsSYAFFV





452
KmSsYAFFV





453
KMsSYAFFV





454
KMSsYAFFV





455
KmsSYAFFVQT





456
KmSsYAFFVQT





457
KMsSYAFFVQT





458
KMSsYAFFVQT





459
KPAsPARRLDL





460
KPDKTLRFsL





461
KPHsPVTGLYL





462
KPLsRVTSL





463
KPPsPGTVL





464
KPPSPGtVL





465
KPRPLsmDL





466
KPRSIsFPSA





467
KPSSLRRVtI





468
KPSsPRGSLLL





469
KQKsLTNLSF





470
KQKSLtNLSF





471
KRAsALLNL





472
KRAsYELEF





473
KRDsFIGTPY





474
KRFsLDFNL





475
KRIsIFLSM





476
KRIsISTSGGSF





477
KRLGsLVDEF





478
KRLsVELTSSL





479
KRLsVELTSSLF





480
KRLsVERIYQK





481
KRMsFVMEY





482
KRNsDLLLL





483
KRPsSEDFVF





484
KRPsSEDFVFL





485
KRPSsEDFVFL





486
KRRtGALVL





487
KRSsISQLL





488
KRVsTFQEF





489
KRVtWIVEF





490
KRYLFRsF





491
KRYsRSLTI





492
KSAsFAFEF





493
KSDGsFIGY





494
KSFsAPATQAY





495
KSGELLAtw





496
KSGEPLStW





497
KSKsIEITF





498
KsLPSDQVmL





499
KsLPSDQVML





500
KSLsIEIGHEV





501
KSLSPsLLGY





502
KSSEEKRLSIsKF





503
KSSsLPRAF





504
KSVtPTKEFL





505
KTDsDSDLQLY





506
KTIsESDLNHSF





507
KTIsPKSTVY





508
KTKsMFFFL





509
KTLsLVKEL





510
KTmsGTFLL





511
KTmSGtFLL





512
KTMSGtFLL





513
KTmsGTFLLRF





514
KTMsGTFLLRF





515
KtMSPSQMIM





516
KTQRVsLLF





517
KtRSLSVEIVY





518
KTRsLSVEIVY





519
KTVsPPIRKGW





520
KTVsSTKLVSF





521
KVDGPRSPsY





522
KVEsPPLEEw





523
KVFsLPTQL





524
KVFsPVIRSSF





525
KVGsFKFIYV





526
KVLswPFLm





527
KVLswPFLM





528
KWPsKRRIPV





529
KYRsVISDIF





530
LAFPsPEKLLR





531
LAsDRCSIHL





532
LEIKEsILSL





533
LEIsPDNSL





534
LEIsVGKSV





535
LEsPTTPLL





536
LESPtTPLL





537
LESPTtPLL





538
LGFEVKsKmV





539
LGFEVKsKMV





540
LGmEVLsGV





541
LGMEVLsGV





542
LIPDHtIRA





543
LLDIIRsL





544
LLDPRSYHtY





545
LLsPKHKY





546
LPAsPRARLSA





547
LPAsPSVSL





548
LPASPsVSL





549
LPDPGsPRL





550
LPEsPRLTL





551
LPFSGPREPsL





552
LPFSsSPSRSA





553
LPFSSsPSRSA





554
LPLsSSHLNVY





555
LPLSsSHLNVY





556
LPLSSsHLNVY





557
LPPVsPLKAA





558
LPRGLsPARQL





559
LPRGSSPsVL





560
LPRPLsPTKL





561
LPRPLSPtKL





562
LPRRLsDSPVF





563
LPRRLSDsPVF





564
LPRsPPLKVL





565
LPRsSRGLL





566
LPRSsRGLL





567
LPRSSsmAAGL





568
LPSARPLsL





569
LPsRLTKc





570
LPTsPLAm





571
LPtSPLAmEY





572
LPtSPLAMEY





573
LPTsPLAmEY





574
LPTsPLAMEY





575
LPVsPGHRKT





576
LPYPVsPKQKY





577
LQHSFsFAGF





578
LQIsPVSSY





579
LSKsSATLw





580
LSPtKLPSI





581
LSRTFKsLF





582
LsSSVIREL





583
LSsSVIREL





584
LTAsQILSR





585
LTDPsSPTISSY





586
LTDPSSPtISSY





587
LTKtLIKL





588
LVAsPRLEK





589
LVREPGsQAcL





590
mIIsPERLDPF





591
MIIsPERLDPF





592
MLPsPNEKL





593
MPFPAHLtY





594
mPHsPTLRV





595
mPHSPtLRV





596
MPHsPTLRV





597
MPHSPtLRV





598
MPKFRMPsL





599
MPQDLRsPA





600
mPREPsATRL





601
mPRQPsATRL





602
mPsPATLSHSL





603
MPsPATLSHSL





604
MPsPFRSSAL





605
mPsPGGRITL





606
MPsPGGRITL





607
MPsPIMHPLIL





608
MPsPLKGQHTL





609
MPsPSTLKKEL





610
mPsPVSPKL





611
mPSPVsPKL





612
MPsPVSPKL





613
MPSPVsPKL





614
MPtSPGVDL





615
MPTsPGVDL





616
mRLsRELQL





617
MSKLINHt





618
mTKSsPLKI





619
NAIsLPTI





620
NAVsPSSGPSL





621
NAWsPVMRAR





622
NHVtPPNVSL





623
NIPsFIVRL





624
NLLsPDGKmISV





625
NmDsPGPML





626
NMDsPGPmL





627
NPIHsPSYPL





628
NPIHSPsYPL





629
NPsSPEFFm





630
NPsSPEFFM





631
NPSsPEFFm





632
NPSsPEFFM





633
NQGsPFKSAL





634
NREsFQIFL





635
NRFsGGFGARDY





636
NRFsPKASL





637
NRHsLPFSL





638
NRHsLVEKL





639
NRLsLLVQK





640
NRMsRRIVL





641
NRSLHINNIsPGNTIS





642
NRSsPVHII





643
NSISSVVsR





644
NSLsPRSSL





645
NSVsPSESL





646
NVLsPLPSQ





647
NVLsPLPSQAM





648
NVMKRKFsL





649
PEFPLsPPKK





650
PEVsPRPAL





651
PIFSRLsI





652
PVSKPLsL





653
QEAsPRPLL





654
QLMtLENKL





655
QLPsPTATSQL





656
QPRNSLPAsPAHQL





657
QPRTPsPLVL





658
QRVPsYDSF





659
QSIsFSGLPSGR





660
QSSsWTRVF





661
QTIsPLSTY





662
QTPDFtPTKY





663
QTPsPRLAL





664
QTRRPsYLEW





665
RAAsIENVL





666
RAAsSPDGFFw





667
RAASsPDGFFw





668
RAAtPLPSL





669
RAAtPTLTTF





670
RAATPtLTTF





671
RAGsFSRFY





672
RAHtPTPGIYm





673
RAHtPTPGIYM





674
RAHTPtPGIYM





675
RALsHADLF





676
RALsLTRAL





677
RANsFVGTAQY





678
RAPsYRTLEL





679
RARsPVLWGW





680
RAsSLNFLNK





681
RASsLNFLNK





682
RAtSNVFAm





683
RAtSNVFAM





684
RATsNVFAm





685
RATsNVFAM





686
RAtSNVFAmF





687
RAtSNVFAMF





688
RATsNVFAmF





689
RATsNVFAMF





690
RATsPLVSLY





691
RAVsPFAKI





692
RAVsPHFDDm





693
RAVsPHFDDM





694
RAYsPLHGGSGSY





695
REAPsPLm





696
REAPsPLM





697
REAsIELPSm





698
REDsLEFSL





699
REDSLEFL





700
REFSGPStPTGTL





701
REFSGPSTPtGTL





702
REImGtPEYL





703
RELsAPARLY





704
RELsGTIKEIL





705
RELsPSSLKm





706
RELsPVSFQY





707
REPsESSPLAL





708
REPSESsPLAL





709
REPsPLPELAL





710
REPsPVRYDNL





711
RERAFsVKF





712
REsPIPIEI





713
REsPRPLQL





714
RESsLGFQL





715
RETNLDsLPL





716
RETsMVHEL





717
RETsPNRIGL





718
REVsPEPIV





719
RFQsmPVRL





720
RFQsMPVRL





721
RHKsDSISL





722
RHLPsPPTL





723
RIGsDPLAY





724
RIIEtPPHRY





725
RIKLGDyHFY





726
RILFsPFFH





727
RILsATTSGIFL





728
RILsDVTHSAV





729
RILsGVVTKm





730
RILsGVVTKM





731
RILsGVVTKMKM





732
RIMsPMRTGNTY





733
RIQsPLNNKL





734
RIRsIEALL





735
RItSLIVHV





736
RITsPVHVSF





737
RIVsPKNSDLK





738
RIWsPTIGR





739
RIWSPtIGR





740
RIYsRIDRLEA





741
RKFsAPGQL





742
RKLsFTESL





743
RKLSFtESL





744
RKLsGDQITL





745
RKLsVALAF





746
RKLsVLLLL





747
RKNsFVmEY





748
RKNsFVEY





749
RKNsLISSL





750
RKSsIIIRm





751
RLAsLFSSL





752
RLAsLMNLGM





753
RLAsYLEKV





754
RLDsELKEL





755
RLDsGHVWKL





756
RLFsKELRc





757
RLFsKSIETL





758
RLFsSFLKR





759
RLIsLSEQNL





760
RLISLsEQNL





761
RLIsQIVSS





762
RLIsQIVSSITA





763
RLIsVVSHL





764
RLKsIEERQLLK





765
RLLQDsVDFSL





766
RLLQDsVDSL





767
RLLsAAENF





768
RLLsEKILGL





769
RLLsIKEAFRL





770
RLLsVNIRV





771
RLNsPPSSIYK





772
RLPLPsPAL





773
RLPsDPFTHL





774
RLPsPTSPFSSL





775
RLPSsTLKR





776
RLPtVLLKL





777
RLQHSFsF





778
RLRsSVPGV





779
RLRSsVPGV





780
RLRsYEDmI





781
RLsPVPVPR





782
RLsSVSVTY





783
RLSsVSVTY





784
RLWtPPEDYRL





785
RLYKsEPEL





786
RLYsVSYLL





787
RmIsHSELRKL





788
RMIsHSELRKL





789
RMIsKLEAQV





790
RmKsPFGSSF





791
RMKsPFGSSF





792
RmLsLRDQRL





793
RmYsFDDVL





794
RNAsLERVL





795
RPADSAQLLsL





796
RPARsVPSIAA





797
RPAsPALLL





798
RPAsPLMI





799
RPASPsLQL





800
RPFHGISTVsLPNSL





801
RPFsKPEIAL





802
RPFsREMDL





803
RPHLSGRKLsL





804
RPHtPTPGI





805
RPHtPTPGIYm





806
RPHTPtPGIYM





807
RPIsPRIGA





808
RPIsVIGGVS





809
RPItPVYTV





810
RPItPVYTVA





811
RPKLHHSLsF





812
RPKPSSsPVI





813
RPKPSsSPVIF





814
RPKPSSsPVIF





815
RPKPsSSPVIFA





816
RPKPSsSPVIFA





817
RPKPSSsPVIFA





818
RPKsTPELAF





819
RPKtPPPAP





820
RPLsKQLSA





821
RPLsLIQGPP





822
RPLsPFYL





823
RPLsPFYLSA





824
RPLsPGALQL





825
RPLsPILHIV





826
RPLsPKPSSPG





827
RPLsPKPSSPGSVL





828
RPLSPKPsSPGSVL





829
RPLsPTRLQPAL





830
RPLtPRTPA





831
RPNsLVGITSA





832
RPNSPsPTAL





833
RPNsSALETL





834
RPNSALETL





835
RPPsPGLRGLL





836
RPQESRsLSPSHL





837
RPQESRSLsPSHL





838
RPQsPPAEAVI





839
RPQtPKEEAQAL





840
RPRAFsHSGVHSL





841
RPRAFsIASSL





842
RPREVtVSL





843
RPRFMsSPVL





844
RPRFMSsPVL





845
RPRGPsPLVTm





846
RPRGPsPLVTM





847
RPRLQHsFSF





848
RPRLQHSFsF





849
RPRPSsVLRTL





850
RPRPVsPSSLLDTAI





851
RPRSIsVEEF





852
RPRSLSsPTVTL





853
RPRsPNmQDL





854
RPRsPPEPLRV





855
RPRSPtGPSNSF





856
RPRtLRTRL





857
RPsSAPDLm





858
RPsSAPDLM





859
RPSsAPDLm





860
RPSsAPDLM





861
RPsSGFYEL





862
RPsSGQDLF





863
RPSsGQDLF





864
RPSsLRQYL





865
RPSsPLIDIKP





866
RPsSPVHVAF





867
RPSsPVHVAF





868
RPSsPVTVTAL





869
RPSsRVALmVL





870
RPSsRVALMVL





871
RPStPHTITL





872
RPsTPTINVL





873
RPStPTINVL





874
RPSTPtINVL





875
RPtSFADEL





876
RPTsISWDGL





877
RPTSIsWDGL





878
RPTsPRLLTL





879
RPVDPRRRsL





880
RPVsEMFSL





881
RPVsMDARIQV





882
RPVsPGKDITA





883
RPVStDFAQY





884
RPVtPITNF





885
RPVtPPRTA





886
RPwsNSRGL





887
RPwsPAVSA





888
RPYPsPGAVL





889
RQAsIELPSMA





890
RQAsIELPSmAV





891
RQAsIELPSmAVA





892
RQAsIELPSmAVAST





893
RQAsIELPSMAVAST





894
RQASLsISV





895
RQFDEESLEsF





896
RQFTSSSsI





897
RQHFsPLSL





898
RQIQPsPPwSY





899
RQIQPsPPWSY





900
RQIsIRGIVGV





901
RQIsISEPQA





902
RQIsISEPQAF





903
RQIsISEPQAFL





904
RQIsISEPQAFLF





905
RQIsPEEFEY





906
RQKsPLFQFA





907
RQPsEEEII





908
RQPsEEEIIKL





909
RQPsWDPSPV





910
RQRSLsTSGESLY





911
RQVsEDPDIDSL





912
RRAsLSDIGF





913
RRFRFPsGAEL





914
RRFsDFLGL





915
RRFSFsGNTL





916
RRFsGLLN





917
RRFsGLLNc





918
RRFsGLLNC





919
RRFsGLSAEL





920
RRFsLDTDY





921
RRFsPPRRML





922
RRFsVTLRL





923
RRFtEIYEF





924
RRFtPPSTAL





925
RRGsFDA





926
RRGsFDAT





927
RRGsFDATG





928
RRGsFDATGSG





929
RRGsFDATGSGF





930
RRGsFDATGSGFSM





931
RRGsFDATGSGFSmTF





932
RRGsFDATGSGFSMTF





933
RRGsFEVTLL





934
RRGsGPEIFTF





935
RRGsPEMPFY





936
RRIDIsPSTFRK





937
RRIDISPsTLRK





938
RRISLtKRL





939
RRLDRRwtL





940
RRLDRRWtL





941
RRLsFQAEYW





942
RRLsLFLVL





943
RRLsVLVDDY





944
RRMsVGDRAG





945
RRMsVGDRAGSLPNY





946
RRNsLRIIF





947
RRPsQNAISFF





948
RRPtLTTFF





949
RRsDSLLSF





950
RRSDsLLSF





951
RRSIIsPNF





952
RRsSFSMEEGDVL





953
RRSsFSMEEGDVL





954
RRsSIPITV





955
RRSsISSWL





956
RRsSLLSLm





957
RRsSLLSLM





958
RRSsLLSLm





959
RRsSYLLAI





960
RRSsYLLAI





961
RRsTGVSFW





962
RRStGVSFW





963
RRTsIHDFL





964
RRVsLSEIGF





965
RRVsSNGIFDL





966
RRVSsNGIFDL





967
RRYsDFAKL





968
RSELLsFIK





969
RSFsADNFIGIQR





970
RSFsGLIKR





971
RSFsMHDLTTI





972
RSFsPKSPLEL





973
RSFsPTmKV





974
RSFSPtMKV





975
RSFtPLSI





976
RSFtPLSILK





977
RSHsPPLKL





978
RSIRDsGYID





979
RSIRDsGYIDcw





980
RSIRDsGYIDcW





981
RSISAsDLTF





982
RSIsNEGLTL





983
RSIsPLLF





984
RSIsPWLAR





985
RSIsQSSTDSY





986
RSIsSLLRF





987
RSIsTPTcL





988
RSKsVIEQV





989
RSKsVIEQVSW





990
RSLsFSDEM





991
RSLsPFRRH





992
RSLsPIIGKDVL





993
RSLsPILPGR





994
RSLsPmSGL





995
RSLsPMSGL





996
RSLsPSSNSAF





997
RsLSQELVGV





998
RsLSVEIVY





999
RSLsVGSEF





1000
RSLsVPVDL





1001
RSLsVPVDLSRW





1002
RSLtHPPTI





1003
RSmDSVLtL





1004
RSMDSVLtL





1005
RSNsPLPSI





1006
RSPsFGEDYY





1007
RSPsQDFSF





1008
RSQsLPNSL





1009
RSRsAPPNLW





1010
RSRsFDYNY





1011
RSRsFDYNYR





1012
RSRsFSGLIKR





1013
RSRSFsGLIKR





1014
RSRsPFSTTR





1015
RSRsPLELEPEAK





1016
RSRsPLGFYV





1017
RSRsPLLKF





1018
RSRsPSDSAAYF





1019
RSRsVPVSF





1020
RSSsFKDFAK





1021
RSSsFSDTL





1022
RSsSFVLPK





1023
RSSsFVLPK





1024
RsSSFVLPKL





1025
RSsSFVLPKL





1026
RSSsFVLPKL





1027
RsSSLSDFSw





1028
RsSSLSDFSW





1029
RSsSLSDFSw





1030
RSsSLSDFSW





1031
RSSsLSDFSw





1032
RSSsLSDFSW





1033
RsSSPFLSK





1034
RSsSPFLSK





1035
RSSsPPILTK





1036
RSsSTELLSHY





1037
RSSsTELLSHY





1038
RSSsWGRTY





1039
RSStPLPTI





1040
RsTSLSLKY





1041
RStSLSLKY





1042
RSTsLSLKY





1043
RSVsFKLLERW





1044
RSVsPVQDL





1045
RSVsVATGL





1046
RSWsPPPEVSR





1047
RSYRTDIsM





1048
RTAsPPALPK





1049
RTFsDESNVL





1050
RtFSLDTIL





1051
RTFsLDTILSSY





1052
RTFSPtYGL





1053
RtHSLLLLL





1054
RtISAQDTLAY





1055
RTIsAQDTLAY





1056
RTIsNPEVVmK





1057
RTIsNPEVVMK





1058
RTKsFLNYY





1059
RTLsESFSRIALK





1060
RTLsGSILDVY





1061
RtmSEAALVRK





1062
RtMSEAALVRK





1063
RTmsPIQVL





1064
RTMsPIQVL





1065
RTPsPARPAL





1066
RTRLsPPRA





1067
RTVsPAHVL





1068
RTYsFTSAm





1069
RTYsFTSAM





1070
RVASPtSGV





1071
RVDSLVsL





1072
RVDsTTcLF





1073
RVDStTcLF





1074
RVDSTtcLF





1075
RVIsLEDFMEK





1076
RVKTPtSQSY





1077
RVKVDGPRsPSY





1078
RVKVDGPRSPsY





1079
RVLsPLmSR





1080
RVLsPLMSR





1081
RVPsINQKI





1082
RVRsFLRGLP





1083
RVRsPGTGAF





1084
RVsSLTLHL





1085
RVSsLTLHL





1086
RVSSLtLHL





1087
RVVLtPLKV





1088
RVVsPGIDL





1089
RVYsLDDIRRY





1090
RVYsRFEVF





1091
RVYYsPPVARR





1092
RWNsKENLL





1093
RYARYsPRQR





1094
RYDsRTTIF





1095
RYFKtPRKF





1096
RYHsLAPmYY





1097
RYHsLAPMYY





1098
RYtNRVVTL





1099
SAFsSRGSLSL





1100
sAISPTPEI





1101
SAIsPTPEI





1102
SAYGGLTsPGLSY





1103
SEAsLASAL





1104
SEFKAmDsI





1105
SEFsDVDKL





1106
SEIsPIKGSVR





1107
SELRsPRISY





1108
SELtPSESL





1109
SELTPsESL





1110
SEsSIKKKFL





1111
SESsIKKKFL





1112
SFDsREASF





1113
SFLsQDESHDHSF





1114
sGEGDFLAEGGGVR





1115
SGFRsPHLw





1116
SGFRsPHLW





1117
SIDIsQDKL





1118
sIDSPKSYI





1119
SIFRtPISK





1120
SIIKEKtV





1121
SIIsPKVKMAL





1122
SIIsPNFSF





1123
SILsRTPSV





1124
sIPSLVDGF





1125
SIPsLVDGF





1126
SIPTVsGQI





1127
SISsIDREL





1128
SISsmEVNV





1129
SIsTLVTL





1130
SIStLVTL





1131
SItSLEAII





1132
SIVsPRKLPAL





1133
SKMAFLtRVA





1134
SLAsKVTRL





1135
SLAsLLAKV





1136
SLDsPGPEKmAL





1137
SLDsPGPEKMAL





1138
SLFGsPVAK





1139
SLFHtPKFV





1140
SLFSsEESNLGA





1141
SLLsELQHA





1142
SLLsLSATV





1143
SLLsVSHAL





1144
SLLtPVRLPSI





1145
SLmsGTLESL





1146
SLmSGtLESL





1147
SLMSGtLESL





1148
SLSsERYYL





1149
SLsSLRAHLEY





1150
SLSsLRAHLEY





1151
SmKsPLYLVSR





1152
SMKsPLYLVSR





1153
SPAARSLsL





1154
SPAsPLKEL





1155
SPDIsPPIFRR





1156
SPFKRQLs





1157
SPFLSKRsL





1158
SPFSSRsPSL





1159
SPGsPWKTKL





1160
sPHSPFYQL





1161
SPHsPFYQL





1162
SPIsDEEERL





1163
SPIsPRTQDAL





1164
SPIsPTRQDAL





1165
SPITSsPPKW





1166
SPKPPtRSP





1167
SPKPPTRsP





1168
SPPsPARWSL





1169
SPRAGsPF





1170
SPRAGsPFSPPPSSSS



L





1171
SPRLVsRSSSVL





1172
SPRPPNSPsI





1173
SPRPPNsPSISI





1174
SPRPtSAPAI





1175
SPRPTsAPAI





1176
SPRRPsRVSEF





1177
SPRRPsRVSEFL





1178
sPRSPISPEL





1179
SPRsPISPEL





1180
sPRSPSTTYL





1181
SPRsPTTTL





1182
SPRsPVNKTTL





1183
sPRSPVPTTL





1184
SPRsPVPTTL





1185
sPRTPPPLTV





1186
SPRtPPPLTV





1187
SPRTPtPFKHAL





1188
SPRtPVSPVKF





1189
SPsPLPVAL





1190
SPsPmDPHM





1191
SPsPMDPHm





1192
SPsPMDPHM





1193
SPtSPDYSL





1194
SPtSPFSSL





1195
SPTsPFSSL





1196
SPVNKVRRVsF





1197
SPVsPKSLAF





1198
SPVsPmKEL





1199
SQDsPIFm





1200
SQDsPIFM





1201
SQILRTPsL





1202
SRFHsPSTTW





1203
SRFsGGFGA





1204
SRFsGGFGARDY





1205
SRHsGPFFTF





1206
SRKEsYSVYVY





1207
SRKsFVFEL





1208
SRLsLRR





1209
SRLsLRRSL





1210
SRPSmsPTPL





1211
SRPSMsPTPL





1212
SRRsIFEMY





1213
SRSsPLKL





1214
SSIsPSTLTLK





1215
SSLsGEELVTK





1216
SSLSsPLNPK





1217
SSSsPFKFK





1218
STAsAITPSVSR





1219
STGGGTVIsR





1220
STsLEKNNV





1221
SVFsPSFGLK





1222
SVIsDDSVL





1223
SVIsGISSR





1224
SVISsPLLK





1225
SVLsPLLNK





1226
SVLsPTSWEK





1227
SVLsYTSVR





1228
SVLtPLLLR





1229
SVPEFPLsPPKK





1230
SVQsDQGYISR





1231
SVSsLEVHF





1232
SVTsPIKmK





1233
SVIsPIKMK





1234
SVVsFDKVKEPR





1235
SVVsGSEMSGKY





1236
SVYsPSGPVNR





1237
SVYSPsGPVNR





1238
SYPsPVPTSF





1239
SYVTTSTRTYsLG





1240
SYYsPSIGFSY





1241
TAIsPPLSV





1242
TELPKRLsL





1243
TESsPGSRQIQLw





1244
TESsPGSRQIQLW





1245
TEVsPSRTI





1246
THALPEsPRL





1247
THDsPFcL





1248
THIsPNAIF





1249
THIsPNAIFKA





1250
TIFsPEGRLY





1251
TImsPAVLK





1252
TIMsPAVLK





1253
TIRSPtTVL





1254
TLAsPSVFK





1255
TLLAsPmLK





1256
TLLsAAHEVEL





1257
TLLsPKHKY





1258
TLPsPDKLPGF





1259
TLSCPVtEVI





1260
TLsSIRHMI





1261
TLSsIRHmI





1262
TLSsIRHMI





1263
TLYPRSFsV





1264
TmFLRETsL





1265
TMFLREtSL





1266
TMFLRETsL





1267
TmLsPREKIFYY





1268
TMLsPREKIFYY





1269
TPAGSARGsPTRPNPP





1270
TPHtPKSLL





1271
TPIsPGRASGmTTL





1272
TPIsPGRASGMTTL





1273
tPPSSEKLVSVM





1274
TPQPsKDTLL





1275
TPsPARPAL





1276
TPVsPVKF





1277
TQRKFsLQF





1278
TRDsLLIHL





1279
TSEtPQPPR





1280
TSIsPALAR





1281
TSVGsPSNTIGR





1282
TSYNSISSVVsR





1283
TTEVIRKGsITEY





1284
tTGSPTEFL





1285
TtGSPTEFL





1286
TTGsPTEFL





1287
TVFsPDGHLF





1288
TVFSPtLPAA





1289
TVFsPTLPAAR





1290
TVFtPVEEK





1291
TVKQKYLsF





1292
TVNsPAIYK





1293
TVNsPAIYKF





1294
TVStPPPFQGR





1295
TVsTVGISI





1296
TVVsPRALEL





1297
TVYSsEEAELLK





1298
TYDDRAYSsF





1299
TYVsSFYHAF





1300
VAKRNsLKELW





1301
VARsPLKEF





1302
VEHsPFSSF





1303
VELsPARSw





1304
VELsPARSW





1305
VELsPLKGSVSW





1306
VETsFRKLSF





1307
VETSFRKLsF





1308
VIDsQELSK





1309
VIKsPSWQR





1310
VImsIRTKL





1311
VIMsIRTKL





1312
VLAsPLKTGR





1313
VLFSsPPQm





1314
VLGsQEALHPV





1315
VLPSQVYsL





1316
VmDsPVHL





1317
VmFRtPLASV





1318
VPFKRLsVVF





1319
VPKGPIHsPVEL





1320
VPKKPPPsP





1321
VPNEEDPsL





1322
VPRsPFKVKVL





1323
VPRsPVIKI





1324
VPRtPVGKF





1325
VPSsPLRKA





1326
VPTsPKGRLL





1327
VRKsRAWVL





1328
VRTPSVQsL





1329
VSFsPTDHSL





1330
VSSsPRELL





1331
VVSsPKLAPK





1332
VYIPmsPGAHHF





1333
VYIPMsPGAHHF





1334
VYLPTHtSL





1335
VYLPTHTsL





1336
VYLPTHtSLL





1337
VYLPTHTsLL





1338
VYTsVQAQY





1339
WEDRPStPTIL





1340
WEFGKRDsL





1341
WPRsPGRAFL





1342
WVIGsPEILR





1343
YAFsPKIGR





1344
yEKIHLDFL





1345
YEVEPYsPGL





1346
YHLsPRAFL





1347
YILDSsPEKL





1348
YLRsVGDGETV





1349
YLVsPITGEKI





1350
YPDPHsPFA





1351
YPFLDsPNKYSL





1352
YPSFRRSsL





1353
YPtPYPDEL





1354
YQLsPTKLPSIN





1355
YQRPFSPsAY





1356
YQYsDQGIDY





1357
YRLsPEPTPL





1358
YRPsYSYDY





1359
YRPsYSYDYEFD





1360
YRYDGQHFsL





1361
YRYsLEKAL





1362
YSLDsPGPEKmAL





1363
YSLDsPGPEKMAL





1364
YSLsPSKSYKY





1365
YSmsPGAMR





1366
YSMsPGAmR





1367
YSMsPGAMR





1368
YVKLTPVsL





1369
YVSsPDPQL





1370
YYFsPSGKKF





1371
yYISPRITF





3921
DIAsLVGHEF





3922
DIVsEYTHY





3923
DSADLPPPsAL





3924
DVIDsQELSKVSREF





3925
ETRSPsPISI





3926
FKmIRSQsL





3927
GAVsPGALR





3928
GLPsPRGPGL





3929
GRILsGVVTK





3930
GRMIRAEsGPDLRY





3931
GRmIRAEsGPDLRY





3932
HPDGtPPKL





3933
HPHLRKVsV





3934
HRRIDIsPSTL





3935
KAsSLISLL





3936
KASsLISLL





3937
KIPsAVSTVSM





3938
KRFsMVVQDGIVK





3939
KRFsmVVQDGIVK





3940
KRFStEEFVLL





3941
KRIsISTS





3942
KRIsISTSG





3943
KRIsISTSGG





3944
KRLsLDSSLVEY





3945
KRLsLPADIRL





3946
KRTsKYFSL





3947
LPRsSSMAAGL





3948
LPRSsSMAAGL





3949
LQHsFSFAGF





3950
LtSKLSTKD





3951
NPTMLRTHsL





3952
NRsSPVHII





3953
QVLPKtVKLF





3954
RLPSPtSPFSSL





3955
RPKLHHsLSF





3956
RPRsDSLIL





3957
RQPswDPSPV





3958
RRAsAPLPGL





3959
RRASLsEIG





3960
RRAsLSEIG





3961
RRFsADEQFF





3962
RRFsFSANFY





3963
RRFsPPSSSL





3964
RRIDIsPS





3965
RRIsIVENcF





3966
RRLPIFsRLSI





3967
RRLsAIFLRL





3968
RRLsFLVSYI





3969
RRLsFTLERL





3970
RRLsIEGNIAV





3971
RRLsPPTLL





3972
RSFSPtmKV





3973
RSsSFTFHI





3974
RSSsFTFHI





3975
RtAATEVSL





3976
RVDsTTCLF





3977
RVDsTTcLFP





3978
RVPsEHPYL





3979
SAITPSVSRTsF





3980
SEGsEPALLH





3981
SIAsPDVKLNL





3982
SIKsDVPVY





3983
SLALtPPQA





3984
SLKsRLR





3985
SLPsPHPVRY





3986
SPRPSPVPKPsPPL





3987
SRFsSGGA





3988
SRIVRTPsL





3989
SRTSFTSVsR





3990
TMPTsLPNL





3991
TRLsPIAPAPGF





3992
TSNsQKYmSF





3993
TSTSRYLsL





3994
VKTsGSSDRL





3995
NIKsPALAF





3996
LsPRAVSTTF





4149
AHDPSGMFRSQsF





4150
RVAsPAYSL





4151
RRWtLGGMVNR





4152
SIPSTLVsF





4153
RRGsYPFIDF





4154
LtLDQAYSY





4155
SPPsPVEREm





4156
SPPsPVEREM





4157
LYVLsALLI





4158
RPRsLSSPTV





4159
LPIFNRIsV





4160
IPRYHSQsPSm





4161
SPLVRRPsL





4162
EAPKVSRsL





4163
SLDSPsYVLY





4164
REYsPPYAP





4165
YGYEGSEsI





4166
RPSsLPLDF





4167
RPsSLPLDF





4168
TPItPLKDGF





4169
KRFsFKKSFKL





4170
KRNsRLGFLY





4171
RRAsAILPGVL





‘s’, ‘t’, and ‘y’ stand for phosphoserine, phosphothreonine, and phosphotyrosine, respectively.


‘m’ stands for oxidized methionine.


‘w’ stands for oxidized tryptophan.


‘c’ stands for cysteinylated cysteine.






Accordingly, in certain embodiments, the instant disclosure provides an antigenic polypeptide comprising an MHC-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171. In certain embodiments, the amino acid sequence of the MHC-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171. In certain embodiments, the amino acid sequence of the antigenic polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171. In certain embodiments, the antigenic polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171.


In certain embodiments, the MHC-binding peptides disclosed herein are 8 to 50 amino acids, (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids) in length.


In certain embodiments, the antigenic peptides disclosed herein are 8 to 100 amino acids, (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids) in length. In certain embodiments, an antigenic peptide is 8 to 50 amino acids in length.


In certain embodiments, the antigenic peptides disclosed herein are less than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length.


In certain embodiments, the amino acid sequence of the antigenic polypeptides disclosed herein does not comprise more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 contiguous amino acids of a protein (e.g., a naturally occurring protein) that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 98-1371, 3921-3996, and 4149-4171.


In another aspect, the instant disclosure provides an antigenic polypeptide comprising a tumor-associated MHC-binding peptide and an HSP-binding peptide. Exemplary HSP-binding peptides are set forth in Table 2 herein. Exemplary antigenic polypeptides comprising HSP-binding peptides are set forth in Table 3 and Table 4 herein.









TABLE 2







Amino acid sequences of exemplary HSP-


binding peptides, linkers, and HSPs











SEQ ID


Description
Amino Acid Sequence
NO












Consensus
X1X2X3X4X5X6X7, wherein:
1


sequence 1
X1 is omitted, N, F, or Q;




X2 is W, L, or F;




X3 is L or I;




X4 is R, L, or K;




X5 is L, W, or I;




X6 is T, L, F, K, R, or W; and




X7 is W, G, K, or F






Consensus
X1LX2LTX3, wherein:
2


sequence 2
X1 is W or F;




X2 is R or K; and




X3 is W, F, or G






Consensus
NX1LX2LTX3, wherein:
3


sequence 3
X1 is W or F;




X2 is R or K; and




X3 is W, F, or G






Consensus
WLX1LTX2, wherein:
4


sequence 4
X1 is R or K; and




X2 is W or G






Consensus
NWLX1LTX2, wherein:
5


sequence 5
X1 is R or K; and




X2 is W or G






Consensus
NWX1X2X3X4X5, wherein:
6


sequence 6
X1 is L or I;




X2 is L, R, or K;




X3 is L or I;




X4 is T, L, F, K, R, or W; and




X5 is W or K






HSP1
NLLRLTG
7





HSP016
WLRLTW
8





HSP017
NWLRLTW
9





HSP018
WLKLTW
10





HSP019
NWLKLTW
11





HSP020
WLRLTG
12





HSP021
NWLRLTG
13





HSP022
FLRLTF
14





HSP023
NFLRLTF
15





HSP024
WLRLTF
16





HSP025
NWLRLTF
17





HSP040
WLKLTF
18





HSP041
NWLKLTF
19





HSP042
WLKLTG
20





HSP043
NWLKLTG
21





HSP044
FLRLTW
22





HSP045
NFLRLTW
23





HSP046
FLRLTG
24





HSP047
NFLRLTG
25





HSP048
FLKLTW
26





HSP049
NFLKLTW
27





HSP050
FLKLTF
28





HSP051
NFLKLTF
29





HSP103
FLKLTG
30





HSP104
NFLKLTG
31





HSP185
NWLLLTW
32





HSP186
NLLRWTG
33





HSP188
FWLRLTW
34





HSP189
NWLRLLW
35





HSP190
NWLRLFW
36





HSP191
NWLRLKW
37





HSP192
NWIRITW
38





HSP193
QWLRLTW
39





HSP194
NWLKLKW
40





HSP195
NWLKLRW
41





HSP196
NWLKLWK
42





Linkerl
FFRK
43





Linker2
FR
N/A





Consensus
FFRKX1X2X3X4X5X6X7, wherein:
44


sequence 1
X1 is omitted, N, F, or Q;



with N-
X2 is W, L, or F;



terminal
X3 is L or I;



linker
X4 is R, L, or K;




X5 is L, W, or I;




X6 is T, L, F, K, R, or W; and




X7 is W, G, K, or F






Consensus
FFRKX1LX2LTX3, wherein:
45


sequence 2
X1 is W or F;



with N-
X2 is R or K; and



terminal
X3 is W, F, or G



linker







Consensus
FFRKNX1LX2LTX3, wherein:
46


sequence 3
X1 is W or F;



with N-
X2 is R or K; and



terminal
X3 is W, F, or G



linker







Consensus
FFRKWLX1LTX2, wherein:
47


sequence 4
X1 is R or K; and



with N-
X2 is W or G



terminal




linker







Consensus
FFRKNWLX1LTX2, wherein:
48


sequence 5
X1 is R or K; and



with N-
X2 is W or G



terminal




linker







Consensus
FFRKNWX1X2X3X4X5, wherein:
49


sequence 6
X1 is L or I;



with N-
X2 is L, R, or K;



terminal
X3 is L or I;



linker
X4 is T, L, F, K, R, or W; and




X5 is W or K






Linker1-
FFRKNLLRLTG
50


HSP1







Linker2-
FRNLLRLTG
51


HSP1







HSP001
FFRKNLLRLTG
52





HSP003
FFRKNWLLLTW
53





HSP004
FFRKNLLRWTG
54





HSP006
FFRKNWLRLTW
55





HSP012
FFRKNWLKLTW
56





HSP013
FFRKNWIRITW
57





HSP014
FFRKQWLRLTW
58





HSP026
FFRKNWLRLTG
59





HSP027
FFRKNFLRLTF
60





HSP028
FRNWLRLTW
61





HSP029
FRNWLKLTW
62





HSP030
FRNWLRLTG
63





HSP031
FRNFLRLTF
64





HSP055
FFRKNWLKLKW
65





HSP057
FFRKNWLKLRW
66





HSP058
FFRKNWLKLWK
67





Consensus
X1X2X3X4X5X6X7FFRK, wherein:
68


sequence 1
X1 is omitted, N, F, or Q;



with C-
X2 is W, L, or F;



terminal
X3 is L or I;



linker
X4 is R, L, or K;




X5 is L, W, or I;




X6 is T, L, F, K, R, or W; and




X7 is W, G, K, or F






Consensus
X1LX2LTX3FFRK, wherein:
69


sequence 2
X1 is W or F;



with C-
X2 is R or K; and



terminal
X3 is W, F, or G



linker







Consensus
NX2LX2LTX3FFRK, wherein:
70


sequence 3
X1 is W or F;



with C-
X2 is R or K; and



terminal
X3 is W, F, or G



linker







Consensus
WLX2LTX2FFRK, wherein:
71


sequence 4
X1 is R or K; and



with C-
X2 iS W or G



terminal




linker







Consensus
NWLX1LTX2FFRK, wherein:
72


sequence 5
X1 is R or K; and



with C-
X2 iS W or G



terminal




linker







Consensus
NWX2X2X3X4X5FFRK, wherein:
73


sequence 6
X1 is L or I;



with C-
X2 is L, R, or K;



terminal
X3 is L or I;



linker
X4 is T, L, F, K, R, or W; and




X5 is W or K






HSP1-
NLLRLTGFFRK
74


Linker1







HSP1-
NLLRLTGFR
75


Linker2







HSP032
NWLRLTWFFRK
76





HSP033
NWLKLTWFFRK
77





HSP034
NWLRLTGFFRK
78





HSP035
NFLRLTFFFRK
79





HSP036
NWLRLTWFR
80





HSP037
NWLKLTWFR
81





HSP038
NWLRLTGFR
82





HSP039
NFLRLTFFR
83





HSP197
NLLRLTWFFRK
84





HSP198
NRLLLTGFFRK
85





HSP199
NWLLLTWFFRK
86





HSP200
NLLRWTGFFRK
87





HSP201
NRLWLTGFFRK
88





HSP202
FWLRLTWFFRK
89





HSP203
NWLRLLWFFRK
90





HSP204
NWLRLFWFFRK
91





HSP205
NWLRLKWFFRK
92





HSP206
NWIRITWFFRK
93





HSP207
QWLRLTWFFRK
94





HSP208
NWLKLKWFFRK
95





HSP209
NWLKLRWFFRK
96





HSP210
NWLKLWKFFRK
97





rh-Hsc70
SKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFT
3920



DTERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDAVVQSDMKH




WPFMVVNDAGRPKVQVEYKGETKSFYPEEVSSMVLTKMKEIAEA




YLGKTVTNAVVTVPAYFNDSQRQATKDAGTIAGLNVLRIINEPT




AAAIAYGLDKKVGAERNVLIFDLGGGTFDVSILTIEDGIFEVKS




TAGDTHLGGEDFDNRMVNHFIAEFKRKHKKDISENKRAVRRLRT




ACERAKRTLSSSTQASIEIDSLYEGIDFYTSITRARFEELNADL




FRGTLDPVEKALRDAKLDKSQIHDIVLVGGSTRIPKIQKLLQDF




FNGKELNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVT




PLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQ




VYEGERAMTKDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGI




LNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAED




EKQRDKVSSKNSLESYAFNMKATVEDEKLQGKINDEDKQKILDK




CNEIINWLDKNQTAEKEEFEHQQKELEKVCNPIITKLYQSAGGM




PGGMPGGFPGGGAPPSGGASSGPTIEEVD
















TABLE 3







Amino acid sequences of exemplary


antigenic polypeptides








SEQ



ID



NO
Amino Acid Sequence





1372
AELGRLsPRAYFFRKNLLRLTG





1373
AESImsFHIFFRKNLLRLTG





1374
AESIMsFHIFFRKNLLRLTG





1375
AEsLKSLSSELFFRKNLLRLTG





1376
AEtPDIKLFFFRKNLLRLTG





1377
AGFsFVNPKFFRKNLLRLTG





1378
AHDPSGmFRSQsFFFRKNLLRLTG





1379
ALDSGAsLLHLFFRKNLLRLTG





1380
ALmGsPQLVAAFFRKNLLRLTG





1381
ALPPGSYAsLFFRKNLLRLTG





1382
ALPTPALsPSLMFFRKNLLRLTG





1383
ALSsSFLVLFFRKNLLRLTG





1384
ALSSsFLVLFFRKNLLRLTG





1385
ALStPVVEKFFRKNLLRLTG





1386
ALVDGyFRLFFRKNLLRLTG





1387
ALwsPGLAKFFRKNLLRLTG





1388
AmLGSKsPDPYRLFFRKNLLRLTG





1389
APAsPFRQLFFRKNLLRLTG





1390
APAsPLRPLFFRKNLLRLTG





1391
APAsPNHAGVLFFRKNLLRLTG





1392
APFHLtPTLYFFRKNLLRLTG





1393
APKsPSSEWLFFRKNLLRLTG





1394
APRtPPGVTFFFRKNLLRLTG





1395
APsSPDVKLFFRKNLLRLTG





1396
APSsPDVKLFFRKNLLRLTG





1397
APTsPLGHLFFRKNLLRLTG





1398
APVsPRPGLFFRKNLLRLTG





1399
ARFsGFYSmFFRKNLLRLTG





1400
ARFsGFYSMFFRKNLLRLTG





1401
ARFsPKVSLFFRKNLLRLTG





1402
ARGIsPIVFFFRKNLLRLTG





1403
ARYsGSYNDYFFRKNLLRLTG





1404
ASFKAELsYFFRKNLLRLTG





1405
ASFtPTSILKFFRKNLLRLTG





1406
ASFtPTSILKRFFRKNLLRLTG





1407
ASLsPSVSKFFRKNLLRLTG





1408
ATIsPPLQPKFFRKNLLRLTG





1409
AVILPPLsPYFKFFRKNLLRLTG





1410
AVLEyLKIFFRKNLLRLTG





1411
AVNQFsPSLARFFRKNLLRLTG





1412
AVRNFsPTDYYFFRKNLLRLTG





1413
AVRNFSPtDYYFFRKNLLRLTG





1414
AWRRLsRDSGGYFFRKNLLRLTG





1415
AYGGLtSPGLSYFFRKNLLRLTG





1416
AYGGLTsPGLSYFFRKNLLRLTG





1417
AYSsYVHQYFFRKNLLRLTG





1418
CtFGSRQIFFRKNLLRLTG





1419
DFAsPFHERFFRKNLLRLTG





1420
DFHsPIVLGRFFRKNLLRLTG





1421
DIAsPTFRRLFFRKNLLRLTG





1422
DIIRQPsEEEIIKFFRKNLLRLTG





1423
DIKsVFEAFFFRKNLLRLTG





1424
DILsPRLIRFFRKNLLRLTG





1425
DIRRFsLTTLRFFRKNLLRLTG





1426
DIsPPIFRRFFRKNLLRLTG





1427
DLtLKKEKFFFRKNLLRLTG





1428
DMLGLtKPAMPMFFRKNLLRLTG





1429
DNFsPDLRVLRFFRKNLLRLTG





1430
DPFGRPTsFFFRKNLLRLTG





1431
DPLIRWDsYFFRKNLLRLTG





1432
DPSLDLHsLFFRKNLLRLTG





1433
DSDPmLsPRFYFFRKNLLRLTG





1434
DSDPMLsPRFYFFRKNLLRLTG





1435
DSDPmLsPRFYAYFFRKNLLRLTG





1436
DSDPMLsPRFYAYFFRKNLLRLTG





1437
DsGEGDFLAEGGGVRFFRKNLLRLTG





1438
DSKsPLGFYFFRKNLLRLTG





1439
DTIsLASERYFFRKNLLRLTG





1440
DTIsPTLGFFFRKNLLRLTG





1441
DTQSGsLLFIGRFFRKNLLRLTG





1442
DTsSLPTVIMRFFRKNLLRLTG





1443
DTSsLPTVImRFFRKNLLRLTG





1444
DTSsLPTVIMRFFRKNLLRLTG





1445
DTTsLRTLRIFFRKNLLRLTG





1446
DVAsPDGLGRLFFRKNLLRLTG





1447
DVAsPTLRFFRKNLLRLTG





1448
DVAsPTLRRFFRKNLLRLTG





1449
DVAsPTLRRLFFRKNLLRLTG





1450
DVIDsQELSKVFFRKNLLRLTG





1451
DVYSGtPTKVFFRKNLLRLTG





1452
DYSPYFKtIFFRKNLLRLTG





1453
EAsSPVPYLFFRKNLLRLTG





1454
EASsPVPYLFFRKNLLRLTG





1455
EEAPQtPVAFFFRKNLLRLTG





1456
EEDtYEKVFFFRKNLLRLTG





1457
EEFsPRQAQmFFFRKNLLRLTG





1458
EEFsPRQAQMFFFRKNLLRLTG





1459
EEIsPTKFPGLFFRKNLLRLTG





1460
EEIsPTKFPGLYFFRKNLLRLTG





1461
EELsPLALGRFFFRKNLLRLTG





1462
EELsPSTVLYFFRKNLLRLTG





1463
EELSPsTVLYFFRKNLLRLTG





1464
EELSPtAKFFFRKNLLRLTG





1465
EGPEtGYSLFFRKNLLRLTG





1466
EHERSIsPLLFFFRKNLLRLTG





1467
EIVNFsPIARFFRKNLLRLTG





1468
ERLKIRGsLFFRKNLLRLTG





1469
ERVDSLVsLFFRKNLLRLTG





1470
ESFSDyPPLGRFAFFRKNLLRLTG





1471
ESLsPIGDmKVFFRKNLLRLTG





1472
ESLsPIGDMKVFFRKNLLRLTG





1473
ESVYKASLsLFFRKNLLRLTG





1474
ETRRPsYLEWFFRKNLLRLTG





1475
EVIRKGsITEYFFRKNLLRLTG





1476
EVIsQHLVSYFFRKNLLRLTG





1477
EVIsVLQKYFFRKNLLRLTG





1478
EVLERKIsMFFRKNLLRLTG





1479
FAFPGStNSLFFRKNLLRLTG





1480
FAFPGSTNsLFFRKNLLRLTG





1481
FASPtSPPVLFFRKNLLRLTG





1482
FASPTsPPVLFFRKNLLRLTG





1483
FATIKSAsLFFRKNLLRLTG





1484
FATIRTAsLFFRKNLLRLTG





1485
FAVsPIPGRGGVLFFRKNLLRLTG





1486
FAwsPLAGEKFFFRKNLLRLTG





1487
FAWsPLAGEKFFFRKNLLRLTG





1488
FAYsPGGAHGmLFFRKNLLRLTG





1489
FFFtARTSFFFRKNLLRLTG





1490
FGGQRLtLFFRKNLLRLTG





1491
FHGISTVsLFFRKNLLRLTG





1492
FHVtPLKLFFRKNLLRLTG





1493
FIVsPVPESRLFFRKNLLRLTG





1494
FKVsPLTFGRFFRKNLLRLTG





1495
FLDsAYFRLFFRKNLLRLTG





1496
FLDsGTIRGVFFRKNLLRLTG





1497
FLFsPPEVTGRFFRKNLLRLTG





1498
FLKPsTSGDSLFFRKNLLRLTG





1499
FLKPSTsGDSLFFRKNLLRLTG





1500
FLKPSTSGDsLFFRKNLLRLTG





1501
FLNEKARLsYFFRKNLLRLTG





1502
FLsRSIPSLFFRKNLLRLTG





1503
FPDNsDVSSIGRLFFRKNLLRLTG





1504
FPDNSDVSsIGRLFFRKNLLRLTG





1505
FPLMRSKsLFFRKNLLRLTG





1506
FPLsPTKLSQYFFRKNLLRLTG





1507
FPSMPsPRLFFRKNLLRLTG





1508
FQYSKSPsLFFRKNLLRLTG





1509
FRFsPMGVDHMFFRKNLLRLTG





1510
FRPPPLtPEDVGFFFRKNLLRLTG





1511
FRRPDIQYPDAtDEFFRKNLLRLTG





1512
FRRsDDMFTFFFRKNLLRLTG





1513
FRYSGKtEYFFRKNLLRLTG





1514
FSFKKsFKLFFRKNLLRLTG





1515
FSFsPGAGAFRFFRKNLLRLTG





1516
FSLRYsPGmDAYFFRKNLLRLTG





1517
FSLRYsPGMDAYFFRKNLLRLTG





1518
FSRPSMsPTPLDRFFRKNLLRLTG





1519
FSVDsPRIYFFRKNLLRLTG





1520
FTIFRTIsVFFRKNLLRLTG





1521
FtPPVVKRFFRKNLLRLTG





1522
FVLsPIKEPAFFRKNLLRLTG





1523
FVRsPGTGAFFFRKNLLRLTG





1524
FVtTPTAELFFRKNLLRLTG





1525
FVTtPTAELFFRKNLLRLTG





1526
FVTTPtAELFFRKNLLRLTG





1527
FYYsPSGKKFFFRKNLLRLTG





1528
GALsRYLFRFFRKNLLRLTG





1529
GEDPLsPRALFFRKNLLRLTG





1530
GELEsIGELFFFRKNLLRLTG





1531
GEmsPQRFFFFRKNLLRLTG





1532
GEMsPQRFFFFRKNLLRLTG





1533
GEmsPQRFFFFFRKNLLRLTG





1534
GENKsPLLLFFRKNLLRLTG





1535
GEPRAPtPPSGTEVTLFFRKNLLRLT



G





1536
GEPsPPHDILFFRKNLLRLTG





1537
GEtSPRTKITWFFRKNLLRLTG





1538
GETsPRTKITWFFRKNLLRLTG





1539
GEwsASLPHRFFFRKNLLRLTG





1540
GEwSAsLPHRFFFRKNLLRLTG





1541
GEWsASLPHRFFFRKNLLRLTG





1542
GEYsPGTALPFFRKNLLRLTG





1543
GGLTsPGLSYFFRKNLLRLTG





1544
GGSISVQVNSIKFDsEFFRKNLLRLT



G





1545
GHGsPFPSLFFRKNLLRLTG





1546
GIFPGtPLKKFFRKNLLRLTG





1547
GIISsPLTGKFFRKNLLRLTG





1548
GIISSPLtGKFFRKNLLRLTG





1549
GImsPLAKKFFRKNLLRLTG





1550
GLFsPIRSSAFFFRKNLLRLTG





1551
GLLsLSALGSQAHLFFRKNLLRLTG





1552
GLPGGGsPTTFLFFRKNLLRLTG





1553
GLSsLSIHLFFRKNLLRLTG





1554
GLTsPGLSYSLFFRKNLLRLTG





1555
GLtVSIPGLFFRKNLLRLTG





1556
GMAILsLLLKFFRKNLLRLTG





1557
GPGHHHKPGLGEGtPFFRKNLLRLTG





1558
GPLSRVKsLFFRKNLLRLTG





1559
GPLVRQIsLFFRKNLLRLTG





1560
GPRAPSPtKPLFFRKNLLRLTG





1561
GPRsASLLFFRKNLLRLTG





1562
GPRSFtPLSIFFRKNLLRLTG





1563
GPRsPKAWLFFRKNLLRLTG





1564
GPRtPTQPLLFFRKNLLRLTG





1565
GRNsLSSLPTYFFRKNLLRLTG





1566
GRQSPsFKLFFRKNLLRLTG





1567
GSFAsPGRLFFFRKNLLRLTG





1568
GsFRGFPALFFRKNLLRLTG





1569
GSKsPDPYRLFFRKNLLRLTG





1570
GSRsLYNLRFFRKNLLRLTG





1571
GTFPKALsIFFRKNLLRLTG





1572
GtPLSQAIIHQYFFRKNLLRLTG





1573
GTVtPPPRLVKFFRKNLLRLTG





1574
GTYVPSsPTRLAYFFRKNLLRLTG





1575
GVIKsPSWQRFFRKNLLRLTG





1576
GVIsPQELLKFFRKNLLRLTG





1577
GVIsPQELLKKFFRKNLLRLTG





1578
GVLsPDTISSKFFRKNLLRLTG





1579
GVmtPLIKRFFRKNLLRLTG





1580
GVMtPLIKRFFRKNLLRLIG





1581
HEFsSPSHLLFFRKNLLRLTG





1582
HEFSsPSHLLFFRKNLLRLTG





1583
HELsDITELFFRKNLLRLTG





1584
HERSIsPLLFFRKNLLRLTG





1585
HFDsPPHLLFFRKNLLRLTG





1586
HHHKPGLGEGtPFFRKNLLRLTG





1587
HHPGLGEGtPFFRKNLLRLTG





1588
HKIsDYFEYFFRKNLLRLTG





1589
HLLEtTPKSEFFRKNLLRLTG





1590
HLLETtPKSEFFRKNLLRLTG





1591
HLLSPtKGIFFRKNLLRLTG





1592
HLNsLDVQLFFRKNLLRLTG





1593
HLPsPPLTQEVFFRKNLLRLTG





1594
HLSsFTMKLFFRKNLLRLTG





1595
HPIsPYEHLFFRKNLLRLTG





1596
HPIsPYEHLLFFRKNLLRLTG





1597
HPIsSEELLFFRKNLLRLTG





1598
HPISsEELLFFRKNLLRLTG





1599
HPIsSEELLSLKYFFRKNLLRLTG





1600
HPISsEELLSLKYFFRKNLLRLTG





1601
HPRPVPDsPVSVTRLFFRKNLLRLTG





1602
HPRsPNVLSVALFFRKNLLRLTG





1603
HPsLSAPALFFRKNLLRLTG





1604
HPSLsAPALFFRKNLLRLTG





1605
HPTLQAPsLFFRKNLLRLTG





1606
HPYRNsDPVIFFRKNLLRLTG





1607
HQFsLKENwFFRKNLLRLTG





1608
HQGKFLQtFFFRKNLLRLTG





1609
HRAsKVLFLFFRKNLLRLTG





1610
HRDsFSRmSLFFRKNLLRLTG





1611
HRDsFSRMSLFFRKNLLRLTG





1612
HRNsmKVFLFFRKNLLRLTG





1613
HRVsVILKLFFRKNLLRLTG





1614
HSDKRRPPsAELYFFRKNLLRLTG





1615
HSLsLDDIRLYFFRKNLLRLTG





1616
HSVsPDPVLFFRKNLLRLTG





1617
HTIsPLDLAFFRKNLLRLTG





1618
HTIsPLDLAKFFRKNLLRLTG





1619
HTIsPLDLAKLFFRKNLLRLTG





1620
HTIsPSFQLFFRKNLLRLTG





1621
HTISPsFQLFFRKNLLRLTG





1622
HVSLITPtKRFFRKNLLRLTG





1623
HYFsPFRPYFFRKNLLRLTG





1624
HYsSRLGSAIFFFRKNLLRLTG





1625
HYSsRLGSAIFFFRKNLLRLTG





1626
HYSSRLGsAIFFFRKNLLRLTG





1627
IAATKsLSVFFRKNLLRLTG





1628
IEIERILsVFFRKNLLRLTG





1629
IFDLQKTsLFFRKNLLRLTG





1630
IIQsPSSTGLLKFFRKNLLRLTG





1631
ILGPPPPsFHLFFRKNLLRLTG





1632
ILLtDLIIFFRKNLLRLTG





1633
IMKNLQAHyEFFRKNLLRLTG





1634
IPHQRSsLFFRKNLLRLTG





1635
IPKsKFLALFFRKNLLRLTG





1636
IPMtPTSSFFFRKNLLRLTG





1637
IPMTPtSSFFFRKNLLRLTG





1638
IPRPLsLIGFFRKNLLRLTG





1639
IPRsFRHLSFFFRKNLLRLTG





1640
IPsmSHVHLFFRKNLLRLTG





1641
IPsMSHVHLFFRKNLLRLTG





1642
IPsPLQPEmFFRKNLLRLTG





1643
IPsPLQPEMFFRKNLLRLTG





1644
IPVSKPLsLFFRKNLLRLTG





1645
IPVsRDWELFFRKNLLRLTG





1646
IRFGRKPsLFFRKNLLRLTG





1647
IRPsVLGPLFFRKNLLRLTG





1648
IRRsYFEVFFFRKNLLRLTG





1649
IRYSGHsLFFRKNLLRLTG





1650
ISKKLsFLSWFFRKNLLRLTG





1651
ISLDKLVsIFFRKNLLRLTG





1652
IsSLTTLSIFFRKNLLRLTG





1653
ISsLTTLSIFFRKNLLRLTG





1654
ISsSmHSLYFFRKNLLRLIG





1655
ISsSMHSLYFFRKNLLRLTG





1656
ISSsmHSLYFFRKNLLRLTG





1657
ITItPPEKYFFRKNLLRLTG





1658
ITLLsPKHKYFFRKNLLRLTG





1659
ItPPSSEKLVSVmFFRKNLLRLTG





1660
ItPPSSEKLVSVMFFRKNLLRLTG





1661
ITTsPITVRFFRKNLLRLTG





1662
ITTsPITVRKFFRKNLLRLTG





1663
ITYsPKLERFFRKNLLRLTG





1664
IVLPLsLQRFFRKNLLRLTG





1665
IVsSLRLAYFFRKNLLRLTG





1666
IVSsLRLAYFFRKNLLRLTG





1667
IYDsVKVYFFFRKNLLRLTG





1668
IYRSQsPHYFFFRKNLLRLTG





1669
KAFsESGSNLHALFFRKNLLRLTG





1670
KAFsPVRSVRFFRKNLLRLTG





1671
KAFsPVRSVRKFFRKNLLRLTG





1672
KAItPPQQPYFFRKNLLRLTG





1673
KASsPGHPAFFFRKNLLRLTG





1674
KAVsFHLVHFFRKNLLRLTG





1675
KAVsLFLFFRKNLLRLTG





1676
KAYtPVVVTQWFFRKNLLRLTG





1677
KEDsFLQRYFFRKNLLRLTG





1678
KEmSPtRQLFFRKNLLRLTG





1679
KEsEVFYELFFRKNLLRLTG





1680
KEsTLHLVLFFRKNLLRLTG





1681
KEStLHLVLFFRKNLLRLTG





1682
KFLsPAQYLYFFRKNLLRLTG





1683
KFRDLsPPRYFFRKNLLRLTG





1684
KFsLRAAEFFFRKNLLRLTG





1685
KGFsGTFQLFFRKNLLRLTG





1686
KIFERATsFFFRKNLLRLTG





1687
KIFsKQQGKAFQRFFRKNLLRLTG





1688
KIIsIFSGFFRKNLLRLTG





1689
KIIsIFSGTEKFFRKNLLRLTG





1690
KIKsLEEIYLFFRKNLLRLTG





1691
KINsLAHLRFFRKNLLRLTG





1692
KISsFTSLKFFRKNLLRLTG





1693
KISSFtSLKFFRKNLLRLTG





1694
KISSFTsLKFFRKNLLRLTG





1695
KISsLEIKLFFRKNLLRLTG





1696
KKLsLLNGGLFFRKNLLRLTG





1697
KLEGPDVsLFFRKNLLRLTG





1698
KLFHGsLEELFFRKNLLRLTG





1699
KLFPGsPAIYFFRKNLLRLTG





1700
KLHsLIGLGIFFRKNLLRLTG





1701
KLIDIVSsQKVFFRKNLLRLTG





1702
KLKsFTYEYFFRKNLLRLTG





1703
KLLDFGsLSNLFFRKNLLRLTG





1704
KLLEGEESRIsLFFRKNLLRLTG





1705
KLLsPILARYFFRKNLLRLTG





1706
KLLsTALHVFFRKNLLRLTG





1707
KLLsYIQRLFFRKNLLRLTG





1708
KLMsDVEDVSLFFRKNLLRLTG





1709
KLMsLGDIRLFFRKNLLRLTG





1710
KLmsPKADVKLFFRKNLLRLTG





1711
KLMsPVLKQHLFFRKNLLRLTG





1712
KLQEFsKEEFFRKNLLRLTG





1713
KLRIQtDGDKYFFRKNLLRLTG





1714
KLSsGLLPKLFFRKNLLRLTG





1715
KLwtLVSEQTRVFFRKNLLRLTG





1716
KLWtLVSEQTRVFFRKNLLRLTG





1717
KLYRPGsVAYFFRKNLLRLTG





1718
KLYsISSQVFFRKNLLRLTG





1719
KLYsPTSKALFFRKNLLRLTG





1720
KLYSPtSKALFFRKNLLRLTG





1721
KLYTyIQSRFFRKNLLRLTG





1722
KLYTyIQSRFFFRKNLLRLTG





1723
KmDsFLDMQLFFRKNLLRLTG





1724
KMDsFLDmQLFFRKNLLRLTG





1725
KmsSYAFFVFFRKNLLRLTG





1726
KmSsYAFFVFFRKNLLRLTG





1727
KMsSYAFFVFFRKNLLRLTG





1728
KMSsYAFFVFFRKNLLRLTG





1729
KmsSYAFFVQTFFRKNLLRLTG





1730
KmSsYAFFVQTFFRKNLLRLTG





1731
KMsSYAFFVQTFFRKNLLRLTG





1732
KMSsYAFFVQTFFRKNLLRLTG





1733
KPAsPARRLDLFFRKNLLRLTG





1734
KPDKTLRFsLFFRKNLLRLTG





1735
KPHsPVTGLYLFFRKNLLRLTG





1736
KPLsRVTSLFFRKNLLRLTG





1737
KPPsPGTVLFFRKNLLRLTG





1738
KPPSPGtVLFFRKNLLRLTG





1739
KPRPLsmDLFFRKNLLRLTG





1740
KPRSIsFPSAFFRKNLLRLTG





1741
KPSSLRRVtIFFRKNLLRLTG





1742
KPSsPRGSLLLFFRKNLLRLTG





1743
KQKsLTNLSFFFRKNLLRLTG





1744
KQKSLtNLSFFFRKNLLRLTG





1745
KRAsALLNLFFRKNLLRLTG





1746
KRAsYELEFFFRKNLLRLTG





1747
KRDsFIGTPYFFRKNLLRLTG





1748
KRFsLDFNLFFRKNLLRLTG





1749
KRIsIFLSMFFRKNLLRLTG





1750
KRIsISTSGGSFFFRKNLLRLTG





1751
KRLGsLVDEFFFRKNLLRLTG





1752
KRLsVELTSSLFFRKNLLRLTG





1753
KRLsVELTSSLFFFRKNLLRLTG





1754
KRLsVERIYQKFFRKNLLRLTG





1755
KRMsFVMEYFFRKNLLRLTG





1756
KRNsDLLLLFFRKNLLRLTG





1757
KRPsSEDFVFFFRKNLLRLTG





1758
KRPsSEDFVFLFFRKNLLRLTG





1759
KRPSsEDFVFLFFRKNLLRLTG





1760
KRRtGALVLFFRKNLLRLTG





1761
KRSsISQLLFFRKNLLRLTG





1762
KRVsTFQEFFFRKNLLRLTG





1763
KRVtWIVEFFFRKNLLRLTG





1764
KRYLFRsFFFRKNLLRLTG





1765
KRYsRSLTIFFRKNLLRLTG





1766
KSAsFAFEFFFRKNLLRLTG





1767
KSDGsFIGYFFRKNLLRLTG





1768
KSFsAPATQAYFFRKNLLRLTG





1769
KSGELLAtwFFRKNLLRLTG





1770
KSGEPLStWFFRKNLLRLTG





1771
KSKsIEITFFFRKNLLRLTG





1772
KsLPSDQVmLFFRKNLLRLTG





1773
KsLPSDQVMLFFRKNLLRLTG





1774
KSLsIEIGHEVFFRKNLLRLTG





1775
KSLSPsLLGYFFRKNLLRLTG





1776
KSSEEKRLSIsKFFFRKNLLRLTG





1777
KSSsLPRAFFFRKNLLRLTG





1778
KSVtPTKEFLFFRKNLLRLTG





1779
KTDsDSDLQLYFFRKNLLRLTG





1780
KTIsESDLNHSFFFRKNLLRLTG





1781
KTIsPKSTVYFFRKNLLRLTG





1782
KTKsMFFFLFFRKNLLRLTG





1783
KTLsLVKELFFRKNLLRLTG





1784
KTmsGTFLLFFRKNLLRLTG





1785
KTmSGtFLLFFRKNLLRLTG





1786
KTMSGtFLLFFRKNLLRLTG





1787
KTmsGTFLLRFFFRKNLLRLTG





1788
KTMsGTFLLRFFFRKNLLRLTG





1789
KtMSPSQMIMFFRKNLLRLTG





1790
KTQRVsLLFFFRKNLLRLTG





1791
KtRSLSVEIVYFFRKNLLRLTG





1792
KTRsLSVEIVYFFRKNLLRLTG





1793
KTVsPPIRKGWFFRKNLLRLTG





1794
KTVsSTKLVSFFFRKNLLRLTG





1795
KVDGPRSPsYFFRKNLLRLTG





1796
KVEsPPLEEwFFRKNLLRLTG





1797
KVFsLPTQLFFRKNLLRLTG





1798
KVFsPVIRSSFFFRKNLLRLTG





1799
KVGsFKFIYVFFRKNLLRLTG





1800
KVLswPFLmFFRKNLLRLTG





1801
KVLswPFLMFFRKNLLRLTG





1802
KWPsKRRIPVFFRKNLLRLTG





1803
KYRsVISDIFFFRKNLLRLTG





1804
LAFPsPEKLLRFFRKNLLRLTG





1805
LAsDRCSIHLFFRKNLLRLTG





1806
LEIKEsILSLFFRKNLLRLTG





1807
LEIsPDNSLFFRKNLLRLTG





1808
LEIsVGKSVFFRKNLLRLTG





1809
LEsPTTPLLFFRKNLLRLTG





1810
LESPtTPLLFFRKNLLRLTG





1811
LESPTtPLLFFRKNLLRLTG





1812
LGFEVKsKmVFFRKNLLRLTG





1813
LGFEVKsKMVFFRKNLLRLTG





1814
LGmEVLsGVFFRKNLLRLTG





1815
LGMEVLsGVFFRKNLLRLTG





1816
LIPDHtIRAFFRKNLLRLTG





1817
LLDIIRsLFFRKNLLRLTG





1818
LLDPRSYHtYFFRKNLLRLTG





1819
LLsPKHKYFFRKNLLRLTG





1820
LPAsPRARLSAFFRKNLLRLTG





1821
LPAsPSVSLFFRKNLLRLTG





1822
LPASPsVSLFFRKNLLRLTG





1823
LPDPGsPRLFFRKNLLRLTG





1824
LPEsPRLTLFFRKNLLRLTG





1825
LPFSGPREPsLFFRKNLLRLTG





1826
LPFSsSPSRSAFFRKNLLRLTG





1827
LPFSSsPSRSAFFRKNLLRLTG





1828
LPLsSSHLNVYFFRKNLLRLTG





1829
LPLSsSHLNVYFFRKNLLRLTG





1830
LPLSSsHLNVYFFRKNLLRLTG





1831
LPPVsPLKAAFFRKNLLRLTG





1832
LPRGLsPARQLFFRKNLLRLTG





1833
LPRGSSPsVLFFRKNLLRLTG





1834
LPRPLsPTKLFFRKNLLRLTG





1835
LPRPLSPtKLFFRKNLLRLTG





1836
LPRRLsDSPVFFFRKNLLRLTG





1837
LPRRLSDsPVFFFRKNLLRLTG





1838
LPRsPPLKVLFFRKNLLRLTG





1839
LPRsSRGLLFFRKNLLRLTG





1840
LPRSsRGLLFFRKNLLRLTG





1841
LPRSSsmAAGLFFRKNLLRLTG





1842
LPSARPLsLFFRKNLLRLTG





1843
LPsRLTKcFFRKNLLRLTG





1844
LPTsPLAmFFRKNLLRLTG





1845
LPtSPLAmEYFFRKNLLRLTG





1846
LPtSPLAMEYFFRKNLLRLTG





1847
LPTsPLAmEYFFRKNLLRLTG





1848
LPTsPLAMEYFFRKNLLRLTG





1849
LPVsPGHRKTFFRKNLLRLTG





1850
LPYPVsPKQKYFFRKNLLRLTG





1851
LQHSFsFAGFFFRKNLLRLTG





1852
LQIsPVSSYFFRKNLLRLTG





1853
LSKsSATLwFFRKNLLRLTG





1854
LSPtKLPSIFFRKNLLRLTG





1855
LSRTFKsLFFFRKNLLRLTG





1856
LsSSVIRELFFRKNLLRLTG





1857
LSsSVIRELFFRKNLLRLTG





1858
LTAsQILSRFFRKNLLRLTG





1859
LTDPsSPTISSYFFRKNLLRLTG





1860
LTDPSSPtISSYFFRKNLLRLTG





1861
LTKtLIKLFFRKNLLRLTG





1862
LVAsPRLEKFFRKNLLRLTG





1863
LVREPGsQAcLFFRKNLLRLTG





1864
mIIsPERLDPFFFRKNLLRLTG





1865
MIIsPERLDPFFFRKNLLRLTG





1866
MLPsPNEKLFFRKNLLRLTG





1867
MPFPAHLtYFFRKNLLRLTG





1868
mPHsPTLRVFFRKNLLRLTG





1869
mPHSPtLRVFFRKNLLRLTG





1870
MPHsPTLRVFFRKNLLRLTG





1871
MPHSPtLRVFFRKNLLRLTG





1872
MPKFRMPsLFFRKNLLRLTG





1873
MPQDLRsPAFFRKNLLRLTG





1874
mPREPsATRLFFRKNLLRLTG





1875
mPRQPsATRLFFRKNLLRLTG





1876
mPsPATLSHSLFFRKNLLRLTG





1877
MPsPATLSHSLFFRKNLLRLTG





1878
MPsPFRSSALFFRKNLLRLTG





1879
mPsPGGRITLFFRKNLLRLTG





1880
MPsPGGRITLFFRKNLLRLTG





1881
MPsPIMHPLILFFRKNLLRLTG





1882
MPsPLKGQHTLFFRKNLLRLTG





1883
MPsPSTLKKELFFRKNLLRLTG





1884
mPsPVSPKLFFRKNLLRLTG





1885
mPSPVsPKLFFRKNLLRLTG





1886
MPsPVSPKLFFRKNLLRLTG





1887
MPSPVsPKLFFRKNLLRLTG





1888
MPtSPGVDLFFRKNLLRLTG





1889
MPTsPGVDLFFRKNLLRLTG





1890
mRLsRELQLFFRKNLLRLTG





1891
MSKLINHtFFRKNLLRLTG





1892
mTKSsPLKIFFRKNLLRLTG





1893
NAIsLPTIFFRKNLLRLTG





1894
NAVsPSSGPSLFFRKNLLRLTG





1895
NAWsPVMRARFFRKNLLRLTG





1896
NHVtPPNVSLFFRKNLLRLTG





1897
NIPsFIVRLFFRKNLLRLTG





1898
NLLsPDGKmISVFFRKNLLRLTG





1899
NmDsPGPMLFFRKNLLRLTG





1900
NMDsPGPmLFFRKNLLRLTG





1901
NPIHsPSYPLFFRKNLLRLTG





1902
NPIHSPsYPLFFRKNLLRLTG





1903
NPsSPEFFmFFRKNLLRLTG





1904
NPsSPEFFMFFRKNLLRLTG





1905
NPSsPEFFmFFRKNLLRLTG





1906
NPSsPEFFMFFRKNLLRLTG





1907
NQGsPFKSALFFRKNLLRLTG





1908
NREsFQIFLFFRKNLLRLTG





1909
NRFsGGFGARDYFFRKNLLRLTG





1910
NRFsPKASLFFRKNLLRLTG





1911
NRHsLPFSLFFRKNLLRLTG





1912
NRHsLVEKLFFRKNLLRLTG





1913
NRLsLLVQKFFRKNLLRLTG





1914
NRMsRRIVLFFRKNLLRLTG





1915
NRSLHINNIsPGNTISFFRKNLLRLT



G





1916
NRSsPVHIIFFRKNLLRLTG





1917
NSISSVVsRFFRKNLLRLTG





1918
NSLsPRSSLFFRKNLLRLTG





1919
NSVsPSESLFFRKNLLRLTG





1920
NVLsPLPSQFFRKNLLRLTG





1921
NVLsPLPSQAMFFRKNLLRLTG





1922
NVMKRKFsLFFRKNLLRLTG





1923
PEFPLsPPKKFFRKNLLRLTG





1924
PEVsPRPALFFRKNLLRLTG





1925
PIFSRLsIFFRKNLLRLTG





1926
PVSKPLsLFFRKNLLRLTG





1927
QEAsPRPLLFFRKNLLRLTG





1928
QLMtLENKLFFRKNLLRLTG





1929
QLPsPTATSQLFFRKNLLRLTG





1930
QPRNSLPAsPAHQLFFRKNLLRLTG





1931
QPRTPsPLVLFFRKNLLRLTG





1932
QRVPsYDSFFFRKNLLRLTG





1933
QSIsFSGLPSGRFFRKNLLRLTG





1934
QSSsWTRVFFFRKNLLRLTG





1935
QTIsPLSTYFFRKNLLRLTG





1936
QTPDFtPTKYFFRKNLLRLTG





1937
QTPsPRLALFFRKNLLRLTG





1938
QTRRPsYLEWFFRKNLLRLTG





1939
RAAsIENVLFFRKNLLRLTG





1940
RAAsSPDGFFwFFRKNLLRLTG





1941
RASsPDGFFwFFRKNLLRLIG





1942
RAAtPLPSLFFRKNLLRLTG





1943
RAAtPTLTTFFFRKNLLRLTG





1944
RAATPtLTTFFFRKNLLRLTG





1945
RAGsFSRFYFFRKNLLRLTG





1946
RAHtPTPGIYmFFRKNLLRLTG





1947
RAHtPTPGIYMFFRKNLLRLTG





1948
RAHTPtPGIYMFFRKNLLRLTG





1949
RALsHADLFFFRKNLLRLTG





1950
RALsLTRALFFRKNLLRLTG





1951
RNsFVGTAQYFFRKNLLRLTG





1952
RAPsYRTLELFFRKNLLRLTG





1953
RARsPVLWGWFFRKNLLRLTG





1954
RAsSLNFLNKFFRKNLLRLTG





1955
RASsLNFLNKFFRKNLLRLIG





1956
RAtSNVFAmFFRKNLLRLTG





1957
RAtSNVFAMFFRKNLLRLTG





1958
RATsNVFAmFFRKNLLRLTG





1959
RATsNVFAMFFRKNLLRLIG





1960
RAtSNVFAmFFFRKNLLRLTG





1961
RAtSNVFAMFFFRKNLLRLTG





1962
RATsNVFAmFFFRKNLLRLTG





1963
RATsNVFAMFFFRKNLLRLTG





1964
RATsPLVSLYFFRKNLLRLTG





1965
RAVsPFAKIFFRKNLLRLTG





1966
RAVsPHFDDmFFRKNLLRLTG





1967
RAVsPHFDDMFFRKNLLRLTG





1968
RAYsPLHGGSGSYFFRKNLLRLTG





1969
REAPsPLmFFRKNLLRLTG





1970
REAPsPLMFFRKNLLRLTG





1971
REAsIELPSmFFRKNLLRLTG





1972
REDsLEFSLFFRKNLLRLTG





1973
REDSLEFsLFFRKNLLRLTG





1974
REFSGPStPTGTLFFRKNLLRLTG





1975
REFSGPSTPtGTLFFRKNLLRLTG





1976
REImGtPEYLFFRKNLLRLTG





1977
RELsAPARLYFFRKNLLRLTG





1978
RELsGTIKEILFFRKNLLRLTG





1979
RELsPSSLKmFFRKNLLRLTG





1980
RELsPVSFQYFFRKNLLRLTG





1981
REPsESSPLALFFRKNLLRLTG





1982
REPSESsPLALFFRKNLLRLTG





1983
REPsPLPELALFFRKNLLRLTG





1984
REPsPVRYDNLFFRKNLLRLTG





1985
RERAFsVKFFFRKNLLRLTG





1986
REsPIPIEIFFRKNLLRLTG





1987
REsPRPLQLFFRKNLLRLTG





1988
RESsLGFQLFFRKNLLRLTG





1989
RETNLDsLPLFFRKNLLRLTG





1990
RETsMVHELFFRKNLLRLTG





1991
RETsPNRIGLFFRKNLLRLTG





1992
REVsPEPIVFFRKNLLRLTG





1993
RFQsmPVRLFFRKNLLRLTG





1994
RFQsMPVRLFFRKNLLRLTG





1995
RHKsDSISLFFRKNLLRLTG





1996
RHLPsPPTLFFRKNLLRLTG





1997
RIGsDPLAYFFRKNLLRLTG





1998
RIIEtPPHRYFFRKNLLRLTG





1999
RIKLGDyHFYFFRKNLLRLTG





2000
RILFsPFFHFFRKNLLRLTG





2001
RILsATTSGIFLFFRKNLLRLTG





2002
RILsDVTHSAVFFRKNLLRLTG





2003
RILsGVVTKmFFRKNLLRLTG





2004
RILsGVVTKMFFRKNLLRLTG





2005
RILsGVVTKMKMFFRKNLLRLTG





2006
RIMsPMRTGNTYFFRKNLLRLTG





2007
RIQsPLNNKLFFRKNLLRLTG





2008
RIRsIEALLFFRKNLLRLTG





2009
RItSLIVHVFFRKNLLRLTG





2010
RITsPVHVSFFFRKNLLRLTG





2011
RIVsPKNSDLKFFRKNLLRLTG





2012
RIWsPTIGRFFRKNLLRLTG





2013
RIWSPtIGRFFRKNLLRLTG





2014
RIYsRIDRLEAFFRKNLLRLTG





2015
RKFsAPGQLFFRKNLLRLTG





2016
RKLsFTESLFFRKNLLRLTG





2017
RKLSFtESLFFRKNLLRLTG





2018
RKLsGDQITLFFRKNLLRLTG





2019
RKLsVALAFFFRKNLLRLTG





2020
RKLsVLLLLFFRKNLLRLTG





2021
RKNsFVmEYFFRKNLLRLTG





2022
RKNsFVMEYFFRKNLLRLTG





2023
RKNsLISSLFFRKNLLRLTG





2024
RKSsIIIRmFFRKNLLRLTG





2025
RLAsLFSSLFFRKNLLRLTG





2026
RLAsLMNLGMFFRKNLLRLTG





2027
RLAsYLEKVFFRKNLLRLTG





2028
RLDsELKELFFRKNLLRLTG





2029
RLDsGHVWKLFFRKNLLRLTG





2030
RLFsKELRcFFRKNLLRLTG





2031
RLFsKSIETLFFRKNLLRLTG





2032
RLFsSFLKRFFRKNLLRLTG





2033
RLIsLSEQNLFFRKNLLRLTG





2034
RLISLsEQNLFFRKNLLRLTG





2035
RLIsQIVSSFFRKNLLRLTG





2036
RLIsQIVSSITAFFRKNLLRLTG





2037
RLIsVVSHLFFRKNLLRLTG





2038
RLKsIEERQLLKFFRKNLLRLTG





2039
RLLQDsVDFSLFFRKNLLRLTG





2040
RLLQDsVDSLFFRKNLLRLTG





2041
RLLsAAENFFFRKNLLRLIG





2042
RLLsEKILGLFFRKNLLRLTG





2043
RLLsIKEAFRLFFRKNLLRLTG





2044
RLLsVNIRVFFRKNLLRLTG





2045
RLNsPPSSIYKFFRKNLLRLTG





2046
RLPLPsPALFFRKNLLRLTG





2047
RLPsDPFTHLFFRKNLLRLTG





2048
RLPsPTSPFSSLFFRKNLLRLTG





2049
RLPSsTLKRFFRKNLLRLTG





2050
RLPtVLLKLFFRKNLLRLTG





2051
RLQHSFsFFFRKNLLRLTG





2052
RLRsSVPGVFFRKNLLRLTG





2053
RLRSsVPGVFFRKNLLRLTG





2054
RLRsYEDmIFFRKNLLRLTG





2055
RLsPVPVPRFFRKNLLRLTG





2056
RLsSVSVTYFFRKNLLRLTG





2057
RLSsVSVTYFFRKNLLRLTG





2058
RLWtPPEDYRLFFRKNLLRLTG





2059
RLYKsEPELFFRKNLLRLTG





2060
RLYsVSYLLFFRKNLLRLTG





2061
RmIsHSELRKLFFRKNLLRLTG





2062
RMIsHSELRKLFFRKNLLRLTG





2063
RMIsKLEAQVFFRKNLLRLTG





2064
RmKsPFGSSFFFRKNLLRLTG





2065
RMKsPFGSSFFFRKNLLRLTG





2066
RmLsLRDQRLFFRKNLLRLTG





2067
RmYsFDDVLFFRKNLLRLTG





2068
RNAsLERVLFFRKNLLRLTG





2069
RPADSAQLLsLFFRKNLLRLTG





2070
RPARsVPSIAAFFRKNLLRLTG





2071
RPAsPALLLFFRKNLLRLTG





2072
RPAsPLMHIFFRKNLLRLTG





2073
RPASPsLQLFFRKNLLRLTG





2074
RPFHGISTVsLPNSLFFRKNLLRLTG





2075
RPFsKPEIALFFRKNLLRLTG





2076
RPFsREMDLFFRKNLLRLTG





2077
RPHLSGRKLsLFFRKNLLRLTG





2078
RPHtPTPGIFFRKNLLRLTG





2079
RPHtPTPGIYmFFRKNLLRLTG





2080
RPHTPtPGIYMFFRKNLLRLTG





2081
RPIsPRIGAFFRKNLLRLTG





2082
RPIsVIGGVSFFRKNLLRLTG





2083
RPItPVYTVFFRKNLLRLTG





2084
RPItPVYTVAFFRKNLLRLTG





2085
RPKLHHSLsFFFRKNLLRLTG





2086
RPKPSSsPVIFFRKNLLRLTG





2087
RPKPSsSPVIFFFRKNLLRLTG





2088
RPKPSSsPVIFFFRKNLLRLTG





2089
RPKPsSSPVIFAFFRKNLLRLTG





2090
RPKPSsSPVIFAFFRKNLLRLTG





2091
RPKPSSsPVIFAFFRKNLLRLTG





2092
RPKsTPELAFFFRKNLLRLTG





2093
RPKtPPPAPFFRKNLLRLTG





2094
RPLsKQLSAFFRKNLLRLTG





2095
RPLsLIQGPPFFRKNLLRLTG





2096
RPLsPFYLFFRKNLLRLTG





2097
RPLsPFYLSAFFRKNLLRLTG





2098
RPLsPGALQLFFRKNLLRLTG





2099
RPLsPILHIVFFRKNLLRLTG





2100
RPLsPKPSSPGFFRKNLLRLTG





2101
RPLsPKPSSPGSVLFFRKNLLRLTG





2102
RPLSPKPsSPGSVLFFRKNLLRLTG





2103
RPLsPTRLQPALFFRKNLLRLTG





2104
RPLtPRTPAFFRKNLLRLTG





2105
RPNsLVGITSAFFRKNLLRLTG





2106
RPNSPsPTALFFRKNLLRLTG





2107
RPNsSALETLFFRKNLLRLTG





2108
RPNSsALETLFFRKNLLRLTG





2109
RPPsPGLRGLLFFRKNLLRLTG





2110
RPQESRsLSPSHLFFRKNLLRLTG





2111
RPQESRSLsPSHLFFRKNLLRLTG





2112
RPQsPPAEAVIFFRKNLLRLTG





2113
RPQtPKEEAQALFFRKNLLRLTG





2114
RPRAFsHSGVHSLFFRKNLLRLTG





2115
RPRAFsIASSLFFRKNLLRLTG





2116
RPREVtVSLFFRKNLLRLTG





2117
RPRFMsSPVLFFRKNLLRLTG





2118
RPRFMSsPVLFFRKNLLRLTG





2119
RPRGPsPLVTmFFRKNLLRLTG





2120
RPRGPsPLVTMFFRKNLLRLTG





2121
RPRLQHsFSFFFRKNLLRLTG





2122
RPRLQHSFsFFFRKNLLRLTG





2123
RPRPSsVLRTLFFRKNLLRLTG





2124
RPRPVsPSSLLDTAIFFRKNLLRLTG





2125
RPRSIsVEEFFFRKNLLRLTG





2126
RPRSLSsPTVTLFFRKNLLRLTG





2127
RPRsPNmQDLFFRKNLLRLTG





2128
RPRsPPEPLRVFFRKNLLRLTG





2129
RPRSPtGPSNSFFFRKNLLRLTG





2130
RPRtLRTRLFFRKNLLRLTG





2131
RPsSAPDLmFFRKNLLRLTG





2132
RPsSAPDLMFFRKNLLRLTG





2133
RPSsAPDLmFFRKNLLRLTG





2134
RPSsAPDLMFFRKNLLRLTG





2135
RPsSGFYELFFRKNLLRLTG





2136
RPsSGQDLFFFRKNLLRLTG





2137
RPSsGQDLFFFRKNLLRLTG





2138
RPSsLRQYLFFRKNLLRLTG





2139
RPSsPLIDIKPFFRKNLLRLTG





2140
RPsSPVHVAFFFRKNLLRLTG





2141
RPSsPVHVAFFFRKNLLRLTG





2142
RPSsPVTVTALFFRKNLLRLTG





2143
RPSsRVALmVLFFRKNLLRLTG





2144
RPSsRVALMVLFFRKNLLRLTG





2145
RPStPHTITLFFRKNLLRLTG





2146
RPsTPTINVLFFRKNLLRLTG





2147
RPStPTINVLFFRKNLLRLTG





2148
RPSTPtINVLFFRKNLLRLTG





2149
RPtSFADELFFRKNLLRLTG





2150
RPTsISWDGLFFRKNLLRLTG





2151
RPTSIsWDGLFFRKNLLRLTG





2152
RPTsPRLLTLFFRKNLLRLTG





2153
RPVDPRRRsLFFRKNLLRLTG





2154
RPVsEMFSLFFRKNLLRLTG





2155
RPVsMDARIQVFFRKNLLRLTG





2156
RPVsPGKDITAFFRKNLLRLTG





2157
RPVStDFAQYFFRKNLLRLTG





2158
RPVtPITNFFFRKNLLRLTG





2159
RPVtPPRTAFFRKNLLRLTG





2160
RPwsNSRGLFFRKNLLRLTG





2161
RPwsPAVSAFFRKNLLRLTG





2162
RPYPsPGAVLFFRKNLLRLTG





2163
RQAsIELPSMAFFRKNLLRLTG





2164
RQAsIELPSmAVFFRKNLLRLTG





2165
RQAsIELPSmAVAFFRKNLLRLTG





2166
RQAsIELPSmAVASTFFRKNLLRLTG





2167
RQAsIELPSMAVASTFFRKNLLRLTG





2168
RQASLsISVFFRKNLLRLTG





2169
RQFDEESLEsFFFRKNLLRLTG





2170
RQFTSSSsIFFRKNLLRLTG





2171
RQHFsPLSLFFRKNLLRLTG





2172
RQIQPsPPwSYFFRKNLLRLTG





2173
RQIQPsPPWSYFFRKNLLRLTG





2174
RQIsIRGIVGVFFRKNLLRLTG





2175
RQIsISEPQAFFRKNLLRLTG





2176
RQIsISEPQAFFFRKNLLRLTG





2177
RQIsISEPQAFLFFRKNLLRLTG





2178
RQIsISEPQAFLFFFRKNLLRLTG





2179
RQIsPEEFEYFFRKNLLRLTG





2180
RQKsPLFQFAFFRKNLLRLTG





2181
RQPsEEEIIFFRKNLLRLTG





2182
RQPsEEEIIKLFFRKNLLRLTG





2183
RQPsWDPSPVFFRKNLLRLTG





2184
RQRSLsTSGESLYFFRKNLLRLTG





2185
RQVsEDPDIDSLFFRKNLLRLTG





2186
RRAsLSDIGFFFRKNLLRLTG





2187
RRFRFPsGAELFFRKNLLRLTG





2188
RRFsDFLGLFFRKNLLRLTG





2189
RRFSFsGNTLFFRKNLLRLTG





2190
RRFsGLLNFFRKNLLRLTG





2191
RRFsGLLNcFFRKNLLRLTG





2192
RRFsGLLNCFFRKNLLRLTG





2193
RRFsGLSAELFFRKNLLRLTG





2194
RRFsLDTDYFFRKNLLRLTG





2195
RRFsPPRRMLFFRKNLLRLTG





2196
RRFsVTLRLFFRKNLLRLTG





2197
RRFtEIYEFFFRKNLLRLTG





2198
RRFtPPSTALFFRKNLLRLTG





2199
RRGsFDAFFRKNLLRLTG





2200
RRGsFDATFFRKNLLRLTG





2201
RRGsFDATGFFRKNLLRLTG





2202
RRGsFDATGSGFFRKNLLRLTG





2203
RRGsFDATGSGFFFRKNLLRLTG





2204
RRGsFDATGSGFSMFFRKNLLRLTG





2205
RRGsFDATGSGFSmTFFFRKNLLRLT



G





2206
RRGsFDATGSGFSMTFFFRKNLLRLT



G





2207
RRGsFEVTLLFFRKNLLRLTG





2208
RRGsGPEIFTFFFRKNLLRLTG





2209
RRGsPEMPFYFFRKNLLRLTG





2210
RRIDIsPSTFRKFFRKNLLRLTG





2211
RRIDISPsTLRKFFRKNLLRLTG





2212
RRISLtKRLFFRKNLLRLTG





2213
RRLDRRwtLFFRKNLLRLTG





2214
RRLDRRWtLFFRKNLLRLTG





2215
RRLsFQAEYWFFRKNLLRLTG





2216
RRLsLFLVLFFRKNLLRLTG





2217
RRLsVLVDDYFFRKNLLRLTG





2218
RRMsVGDRAGFFRKNLLRLTG





2219
RRMsVGDRAGSLPNYFFRKNLLRLTG





2220
RRNsLRIIFFFRKNLLRLTG





2221
RRPsQNAISFFFFRKNLLRLTG





2222
RRPtLTTFFFFRKNLLRLTG





2223
RRsDSLLSFFFRKNLLRLTG





2224
RRSDsLLSFFFRKNLLRLTG





2225
RRSIIsPNFFFRKNLLRLTG





2226
RRsSFSMEEGDVLFFRKNLLRLTG





2227
RRSsFSMEEGDVLFFRKNLLRLTG





2228
RRsSIPITVFFRKNLLRLTG





2229
RRSsISSWLFFRKNLLRLTG





2230
RRsSLLSLmFFRKNLLRLTG





2231
RRsSLLSLMFFRKNLLRLTG





2232
RRSsLLSLmFFRKNLLRLTG





2233
RRsSYLLAIFFRKNLLRLTG





2234
RRSsYLLAIFFRKNLLRLTG





2235
RRsTGVSFWFFRKNLLRLTG





2236
RRStGVSFWFFRKNLLRLTG





2237
RRTsIHDFLFFRKNLLRLTG





2238
RRVsLSEIGFFFRKNLLRLTG





2239
RRVsSNGIFDLFFRKNLLRLTG





2240
RRVSsNGIFDLFFRKNLLRLTG





2241
RRYsDFAKLFFRKNLLRLTG





2242
RSELLsFIKFFRKNLLRLTG





2243
RSFsADNFIGIQRFFRKNLLRLTG





2244
RSFsGLIKRFFRKNLLRLTG





2245
RSFsMHDLTTIFFRKNLLRLTG





2246
RSFsPKSPLELFFRKNLLRLTG





2247
RSFsPTmKVFFRKNLLRLTG





2248
RSFSPtMKVFFRKNLLRLTG





2249
RSFtPLSIFFRKNLLRLTG





2250
RSFtPLSILKFFRKNLLRLTG





2251
RSHsPPLKLFFRKNLLRLTG





2252
RSIRDsGYIDFFRKNLLRLTG





2253
RSIRDsGYIDcwFFRKNLLRLTG





2254
RSIRDsGYIDcWFFRKNLLRLTG





2255
RSISAsDLTFFFRKNLLRLTG





2256
RSIsNEGLTLFFRKNLLRLTG





2257
RSIsPLLFFFRKNLLRLTG





2258
RSIsPWLARFFRKNLLRLTG





2259
RSIsQSSTDSYFFRKNLLRLTG





2260
RSIsSLLRFFFRKNLLRLTG





2261
RSIsTPTcLFFRKNLLRLTG





2262
RSKsVIEQVFFRKNLLRLTG





2263
RSKsVIEQVSWFFRKNLLRLTG





2264
RSLsFSDEMFFRKNLLRLTG





2265
RSLsPFRRHFFRKNLLRLTG





2266
RSLsPIIGKDVLFFRKNLLRLTG





2267
RSLsPILPGRFFRKNLLRLTG





2268
RSLsPmSGLFFRKNLLRLTG





2269
RSLsPMSGLFFRKNLLRLTG





2270
RSLsPSSNSAFFFRKNLLRLTG





2271
RsLSQELVGVFFRKNLLRLTG





2272
RsLSVEIVYFFRKNLLRLTG





2273
RSLsVGSEFFFRKNLLRLTG





2274
RSLsVPVDLFFRKNLLRLTG





2275
RSLsVPVDLSRWFFRKNLLRLTG





2276
RSLtHPPTIFFRKNLLRLTG





2277
RSmDSVLtLFFRKNLLRLTG





2278
RSMDSVLtLFFRKNLLRLTG





2279
RSNsPLPSIFFRKNLLRLTG





2280
RSPsFGEDYYFFRKNLLRLTG





2281
RSPsQDFSFFFRKNLLRLTG





2282
RSQsLPNSLFFRKNLLRLTG





2283
RSRsAPPNLWFFRKNLLRLTG





2284
RSRsFDYNYFFRKNLLRLTG





2285
RSRsFDYNYRFFRKNLLRLTG





2286
RSRsFSGLIKRFFRKNLLRLTG





2287
RSRSFsGLIKRFFRKNLLRLTG





2288
RSRsPFSTTRFFRKNLLRLTG





2289
RSRsPLELEPEAKFFRKNLLRLTG





2290
RSRsPLGFYVFFRKNLLRLTG





2291
RSRsPLLKFFFRKNLLRLTG





2292
RSRsPSDSAAYFFFRKNLLRLTG





2293
RSRsVPVSFFFRKNLLRLTG





2294
RSSsFKDFAKFFRKNLLRLTG





2295
RSSsFSDTLFFRKNLLRLTG





2296
RSsSFVLPKFFRKNLLRLTG





2297
RSSsFVLPKFFRKNLLRLTG





2298
RsSSFVLPKLFFRKNLLRLTG





2299
RSsSFVLPKLFFRKNLLRLTG





2300
RSSsFVLPKLFFRKNLLRLTG





2301
RsSSLSDFSwFFRKNLLRLTG





2302
RsSSLSDFSWFFRKNLLRLTG





2303
RSsSLSDFSwFFRKNLLRLTG





2304
RSsSLSDFSWFFRKNLLRLTG





2305
RSSsLSDFSwFFRKNLLRLTG





2306
RSSsLSDFSWFFRKNLLRLTG





2307
RsSSPFLSKFFRKNLLRLTG





2308
RSsSPFLSKFFRKNLLRLTG





2309
RSSsPPILTKFFRKNLLRLTG





2310
RSsSTELLSHYFFRKNLLRLTG





2311
RSSsTELLSHYFFRKNLLRLTG





2312
RSSsWGRTYFFRKNLLRLTG





2313
RSStPLPTIFFRKNLLRLTG





2314
RsTSLSLKYFFRKNLLRLTG





2315
RStSLSLKYFFRKNLLRLTG





2316
RSTsLSLKYFFRKNLLRLTG





2317
RSVsFKLLERWFFRKNLLRLTG





2318
RSVsPVQDLFFRKNLLRLTG





2319
RSVsVATGLFFRKNLLRLTG





2320
RSWsPPPEVSRFFRKNLLRLTG





2321
RSYRTDIsMFFRKNLLRLTG





2322
RTAsPPALPKFFRKNLLRLTG





2323
RTFsDESNVLFFRKNLLRLTG





2324
RtFSLDTILFFRKNLLRLTG





2325
RTFsLDTILSSYFFRKNLLRLTG





2326
RTFSPtYGLFFRKNLLRLTG





2327
RtHSLLLLLFFRKNLLRLTG





2328
RtISAQDTLAYFFRKNLLRLTG





2329
RTIsAQDTLAYFFRKNLLRLTG





2330
RTIsNPEVVmKFFRKNLLRLTG





2331
RTIsNPEVVMKFFRKNLLRLIG





2332
RTKsFLNYYFFRKNLLRLTG





2333
RTLsESFSRIALKFFRKNLLRLTG





2334
RTLsGSILDVYFFRKNLLRLTG





2335
RtmSEAALVRKFFRKNLLRLTG





2336
RtMSEAALVRKFFRKNLLRLTG





2337
RTmsPIQVLFFRKNLLRLTG





2338
RTMsPIQVLFFRKNLLRLTG





2339
RTPsPARPALFFRKNLLRLTG





2340
RTRLsPPRAFFRKNLLRLTG





2341
RTVsPAHVLFFRKNLLRLTG





2342
RTYsFTSAmFFRKNLLRLTG





2343
RTYsFTSAMFFRKNLLRLTG





2344
RVASPtSGVFFRKNLLRLTG





2345
RVDSLVsLFFRKNLLRLTG





2346
RVDsTTcLFFFRKNLLRLTG





2347
RVDStTcLFFFRKNLLRLTG





2348
RVDSTtcLFFFRKNLLRLTG





2349
RVIsLEDFMEKFFRKNLLRLTG





2350
RVKTPtSQSYFFRKNLLRLTG





2351
RVKVDGPRsPSYFFRKNLLRLTG





2352
RVKVDGPRSPsYFFRKNLLRLTG





2353
RVLsPLmSRFFRKNLLRLTG





2354
RVLsPLMSRFFRKNLLRLTG





2355
RVPsINQKIFFRKNLLRLTG





2356
RVRsFLRGLPFFRKNLLRLTG





2357
RVRsPGTGAFFFRKNLLRLTG





2358
RVsSLTLHLFFRKNLLRLTG





2359
RVSsLTLHLFFRKNLLRLTG





2360
RVSSLtLHLFFRKNLLRLTG





2361
RVVLtPLKVFFRKNLLRLTG





2362
RVVsPGIDLFFRKNLLRLTG





2363
RVYsLDDIRRYFFRKNLLRLTG





2364
RVYsRFEVFFFRKNLLRLTG





2365
RVYYsPPVARRFFRKNLLRLTG





2366
RWNsKENLLFFRKNLLRLTG





2367
RYARYsPRQRFFRKNLLRLTG





2368
RYDsRTTIFFFRKNLLRLTG





2369
RYFKtPRKFFFRKNLLRLTG





2370
RYHsLAPmYYFFRKNLLRLTG





2371
RYHsLAPMYYFFRKNLLRLTG





2372
RYtNRVVTLFFRKNLLRLTG





2373
SAFsSRGSLSLFFRKNLLRLTG





2374
sAISPTPEIFFRKNLLRLTG





2375
SAIsPTPEIFFRKNLLRLTG





2376
SAYGGLTsPGLSYFFRKNLLRLTG





2377
SEAsLASALFFRKNLLRLTG





2378
SEFKAmDsIFFRKNLLRLTG





2379
SEFsDVDKLFFRKNLLRLTG





2380
SEIsPIKGSVRFFRKNLLRLTG





2381
SELRsPRISYFFRKNLLRLTG





2382
SELtPSESLFFRKNLLRLTG





2383
SELTPsESLFFRKNLLRLTG





2384
SEsSIKKKFLFFRKNLLRLTG





2385
SESsIKKKFLFFRKNLLRLTG





2386
SFDsREASFFFRKNLLRLTG





2387
SFLsQDESHDHSFFFRKNLLRLTG





2388
sGEGDFLAEGGGVRFFRKNLLRLTG





2389
SGFRsPHLwFFRKNLLRLTG





2390
SGFRsPHLWFFRKNLLRLTG





2391
SIDIsQDKLFFRKNLLRLTG





2392
sIDSPKSYIFFRKNLLRLTG





2393
SIFRtPISKFFRKNLLRLTG





2394
SIIKEKtVFFRKNLLRLTG





2395
SIIsPKVKMALFFRKNLLRLTG





2396
SIIsPNFSFFFRKNLLRLTG





2397
SILsRTPSVFFRKNLLRLTG





2398
sIPSLVDGFFFRKNLLRLTG





2399
SIPsLVDGFFFRKNLLRLTG





2400
SIPTVsGQIFFRKNLLRLTG





2401
SISsIDRELFFRKNLLRLTG





2402
SISsmEVNVFFRKNLLRLTG





2403
SIsTLVTLFFRKNLLRLTG





2404
SIStLVTLFFRKNLLRLTG





2405
SItSLEAIIFFRKNLLRLTG





2406
SIVsPRKLPALFFRKNLLRLTG





2407
SKMAFLtRVAFFRKNLLRLTG





2408
SLAsKVTRLFFRKNLLRLTG





2409
SLAsLLAKVFFRKNLLRLTG





2410
SLDsPGPEKmALFFRKNLLRLTG





2411
SLDsPGPEKMALFFRKNLLRLTG





2412
SLFGsPVAKFFRKNLLRLTG





2413
SLFHtPKFVFFRKNLLRLTG





2414
SLFSsEESNLGAFFRKNLLRLTG





2415
SLLsELQHAFFRKNLLRLTG





2416
SLLsLSATVFFRKNLLRLTG





2417
SLLsVSHALFFRKNLLRLTG





2418
SLLtPVRLPSIFFRKNLLRLTG





2419
SLmsGTLESLFFRKNLLRLTG





2420
SLmSGtLESLFFRKNLLRLTG





2421
SLMSGtLESLFFRKNLLRLTG





2422
SLSsERYYLFFRKNLLRLTG





2423
SLsSLRAHLEYFFRKNLLRLTG





2424
SLSsLRAHLEYFFRKNLLRLTG





2425
SmKsPLYLVSRFFRKNLLRLTG





2426
SMKsPLYLVSRFFRKNLLRLTG





2427
SPAARSLsLFFRKNLLRLTG





2428
SPAsPLKELFFRKNLLRLTG





2429
SPDIsPPIFRRFFRKNLLRLTG





2430
SPFKRQLsFFRKNLLRLTG





2431
SPFLSKRsLFFRKNLLRLTG





2432
SPFSSRsPSLFFRKNLLRLTG





2433
SPGsPWKTKLFFRKNLLRLTG





2434
sPHSPFYQLFFRKNLLRLTG





2435
SPHsPFYQLFFRKNLLRLTG





2436
SPIsDEEERLFFRKNLLRLTG





2437
SPIsPRTQDALFFRKNLLRLTG





2438
SPIsPTRQDALFFRKNLLRLTG





2439
SPITSsPPKWFFRKNLLRLTG





2440
SPKPPtRSPFFRKNLLRLTG





2441
SPKPPTRsPFFRKNLLRLTG





2442
SPPsPARWSLFFRKNLLRLTG





2443
SPRAGsPFFFRKNLLRLTG





2444
SPRAGsPFSPPPSSSSLFFRKNLLRL



TG





2445
SPRLVsRSSSVLFFRKNLLRLTG





2446
SPRPPNSPsIFFRKNLLRLTG





2447
SPRPPNsPSISIFFRKNLLRLTG





2448
SPRPtSAPAIFFRKNLLRLTG





2449
SPRPTsAPAIFFRKNLLRLTG





2450
SPRRPsRVSEFFFRKNLLRLTG





2451
SPRRPsRVSEFLFFRKNLLRLTG





2452
sPRSPISPELFFRKNLLRLTG





2453
SPRsPISPELFFRKNLLRLTG





2454
sPRSPSTTYLFFRKNLLRLTG





2455
SPRsPTTTLFFRKNLLRLTG





2456
SPRsPVNKTTLFFRKNLLRLTG





2457
sPRSPVPTTLFFRKNLLRLTG





2458
SPRsPVPTTLFFRKNLLRLTG





2459
sPRTPPPLTVFFRKNLLRLTG





2460
SPRtPPPLTVFFRKNLLRLTG





2461
SPRTPtPFKHALFFRKNLLRLTG





2462
SPRtPVSPVKFFFRKNLLRLTG





2463
SPsPLPVALFFRKNLLRLTG





2464
SPsPmDPHMFFRKNLLRLTG





2465
SPsPMDPHmFFRKNLLRLTG





2466
SPsPMDPHMFFRKNLLRLTG





2467
SPtSPDYSLFFRKNLLRLTG





2468
SPtSPFSSLFFRKNLLRLTG





2469
SPTsPFSSLFFRKNLLRLTG





2470
SPVNKVRRVsFFFRKNLLRLTG





2471
SPVsPKSLAFFFRKNLLRLTG





2472
SPVsPmKELFFRKNLLRLTG





2473
SQDsPIFmFFRKNLLRLTG





2474
SQDsPIFMFFRKNLLRLTG





2475
SQILRTPsLFFRKNLLRLTG





2476
SRFHsPSTTWFFRKNLLRLTG





2477
SRFsGGFGAFFRKNLLRLTG





2478
SRFsGGFGARDYFFRKNLLRLTG





2479
SRHsGPFFTFFFRKNLLRLTG





2480
SRKEsYSVYVYFFRKNLLRLTG





2481
SRKsFVFELFFRKNLLRLTG





2482
SRLsLRRFFRKNLLRLTG





2483
SRLsLRRSLFFRKNLLRLTG





2484
SRPSmsPTPLFFRKNLLRLTG





2485
SRPSMsPTPLFFRKNLLRLTG





2486
SRRsIFEMYFFRKNLLRLTG





2487
SRSsPLKLFFRKNLLRLTG





2488
SSIsPSTLTLKFFRKNLLRLTG





2489
SSLsGEELVTKFFRKNLLRLTG





2490
SSLSsPLNPKFFRKNLLRLTG





2491
SSSsPFKFKFFRKNLLRLTG





2492
STAsAITPSVSRFFRKNLLRLTG





2493
STGGGTVIsRFFRKNLLRLTG





2494
STsLEKNNVFFRKNLLRLTG





2495
SVFsPSFGLKFFRKNLLRLTG





2496
SVIsDDSVLFFRKNLLRLTG





2497
SVIsGISSRFFRKNLLRLTG





2498
SVISsPLLKFFRKNLLRLTG





2499
SVLsPLLNKFFRKNLLRLTG





2500
SVLsPTSWEKFFRKNLLRLTG





2501
SVLsYTSVRFFRKNLLRLTG





2502
SVLtPLLLRFFRKNLLRLTG





2503
SVPEFPLsPPKKFFRKNLLRLTG





2504
SVQsDQGYISRFFRKNLLRLTG





2505
SVSsLEVHFFFRKNLLRLTG





2506
SVTsPIKmKFFRKNLLRLTG





2507
SVTsPIKMKFFRKNLLRLTG





2508
SVVsFDKVKEPRFFRKNLLRLTG





2509
SVVsGSEMSGKYFFRKNLLRLTG





2510
SVYsPSGPVNRFFRKNLLRLTG





2511
SVYSPsGPVNRFFRKNLLRLTG





2512
SYPsPVPTSFFFRKNLLRLTG





2513
SYVTTSTRTYsLGFFRKNLLRLTG





2514
SYYsPSIGFSYFFRKNLLRLTG





2515
TAIsPPLSVFFRKNLLRLTG





2516
TELPKRLsLFFRKNLLRLTG





2517
TESsPGSRQIQLwFFRKNLLRLTG





2518
TESsPGSRQIQLWFFRKNLLRLTG





2519
TEVsPSRTIFFRKNLLRLTG





2520
THALPEsPRLFFRKNLLRLTG





2521
THDsPFcLFFRKNLLRLTG





2522
THIsPNAIFFFRKNLLRLTG





2523
THIsPNAIFKAFFRKNLLRLTG





2524
TIFsPEGRLYFFRKNLLRLTG





2525
TImsPAVLKFFRKNLLRLTG





2526
TIMsPAVLKFFRKNLLRLTG





2527
TIRSPtTVLFFRKNLLRLTG





2528
TLAsPSVFKFFRKNLLRLTG





2529
TLLAsPmLKFFRKNLLRLTG





2530
TLLsAAHEVELFFRKNLLRLTG





2531
TLLsPKHKYFFRKNLLRLTG





2532
TLPsPDKLPGFFFRKNLLRLTG





2533
TLSCPVtEVIFFRKNLLRLTG





2534
TLsSIRHMIFFRKNLLRLTG





2535
TLSsIRHmIFFRKNLLRLTG





2536
TLSsIRHMIFFRKNLLRLTG





2537
TLYPRSFsVFFRKNLLRLTG





2538
TmFLRETsLFFRKNLLRLTG





2539
TMFLREtSLFFRKNLLRLTG





2540
TMFLRETsLFFRKNLLRLTG





2541
TmLsPREKIFYYFFRKNLLRLTG





2542
TMLsPREKIFYYFFRKNLLRLTG





2543
TPAGSARGsPTRPNPPFFRKNLLRLT



G





2544
TPHtPKSLLFFRKNLLRLTG





2545
TPIsPGRASGmTTLFFRKNLLRLTG





2546
TPIsPGRASGMTTLFFRKNLLRLTG





2547
tPPSSEKLVSVMFFRKNLLRLTG





2548
TPQPsKDTLLFFRKNLLRLTG





2549
TPsPARPALFFRKNLLRLTG





2550
TPVsPVKFFFRKNLLRLTG





2551
TQRKFsLQFFFRKNLLRLTG





2552
TRDsLLIHLFFRKNLLRLTG





2553
TSEtPQPPRFFRKNLLRLTG





2554
TSIsPALARFFRKNLLRLTG





2555
TSVGsPSNTIGRFFRKNLLRLTG





2556
TSYNSISSVVsRFFRKNLLRLTG





2557
TTEVIRKGsITEYFFRKNLLRLTG





2558
tTGSPTEFLFFRKNLLRLTG





2559
TtGSPTEFLFFRKNLLRLTG





2560
TTGsPTEFLFFRKNLLRLTG





2561
TVFsPDGHLFFFRKNLLRLTG





2562
TVFSPtLPAAFFRKNLLRLIG





2563
TVFsPTLPAARFFRKNLLRLTG





2564
TVFtPVEEKFFRKNLLRLTG





2565
TVKQKYLsFFFRKNLLRLTG





2566
TVNsPAIYKFFRKNLLRLTG





2567
TVNsPAIYKFFFRKNLLRLTG





2568
TVStPPPFQGRFFRKNLLRLTG





2569
TVsTVGISIFFRKNLLRLTG





2570
TVVsPRALELFFRKNLLRLTG





2571
TVYSsEEAELLKFFRKNLLRLTG





2572
TYDDRAYSsFFFRKNLLRLTG





2573
TYVsSFYHAFFFRKNLLRLTG





2574
VAKRNsLKELWFFRKNLLRLTG





2575
VARsPLKEFFFRKNLLRLTG





2576
VEHsPFSSFFFRKNLLRLTG





2577
VELsPARSwFFRKNLLRLTG





2578
VELsPARSWFFRKNLLRLTG





2579
VELsPLKGSVSWFFRKNLLRLTG





2580
VETsFRKLSFFFRKNLLRLTG





2581
VETSFRKLsFFFRKNLLRLTG





2582
VIDsQELSKFFRKNLLRLTG





2583
VIKsPSWQRFFRKNLLRLTG





2584
VImsIRTKLFFRKNLLRLTG





2585
VIMsIRTKLFFRKNLLRLTG





2586
VLAsPLKTGRFFRKNLLRLTG





2587
VLFSsPPQmFFRKNLLRLTG





2588
VLGsQEALHPVFFRKNLLRLTG





2589
VLPSQVYsLFFRKNLLRLTG





2590
VmDsPVHLFFRKNLLRLTG





2591
VmFRtPLASVFFRKNLLRLTG





2592
VPFKRLsVVFFFRKNLLRLTG





2593
VPKGPIHsPVELFFRKNLLRLTG





2594
VPKKPPPsPFFRKNLLRLTG





2595
VPNEEDPsLFFRKNLLRLTG





2596
VPRsPFKVKVLFFRKNLLRLTG





2597
VPRsPVIKIFFRKNLLRLTG





2598
VPRtPVGKFFFRKNLLRLTG





2599
VPSsPLRKAFFRKNLLRLTG





2600
VPTsPKGRLLFFRKNLLRLTG





2601
VRKsRAWVLFFRKNLLRLTG





2602
VRTPSVQsLFFRKNLLRLTG





2603
VSFsPTDHSLFFRKNLLRLTG





2604
VSSsPRELLFFRKNLLRLTG





2605
VVSsPKLAPKFFRKNLLRLTG





2606
VYIPmsPGAHHFFFRKNLLRLTG





2607
VYIPMsPGAHHFFFRKNLLRLTG





2608
VYLPTHtSLFFRKNLLRLTG





2609
VYLPTHTsLFFRKNLLRLTG





2610
VYLPTHtSLLFFRKNLLRLTG





2611
VYLPTHTsLLFFRKNLLRLTG





2612
VYTsVQAQYFFRKNLLRLTG





2613
WEDRPStPTILFFRKNLLRLTG





2614
WEFGKRDsLFFRKNLLRLTG





2615
WPRsPGRAFLFFRKNLLRLTG





2616
WVIGsPEILRFFRKNLLRLTG





2617
YAFsPKIGRFFRKNLLRLTG





2618
yEKIHLDFLFFRKNLLRLTG





2619
YEVEPYsPGLFFRKNLLRLTG





2620
YHLsPRAFLFFRKNLLRLTG





2621
YILDSsPEKLFFRKNLLRLTG





2622
YLRsVGDGETVFFRKNLLRLTG





2623
YLVsPITGEKIFFRKNLLRLTG





2624
YPDPHsPFAFFRKNLLRLTG





2625
YPFLDsPNKYSLFFRKNLLRLTG





2626
YPSFRRSsLFFRKNLLRLTG





2627
YPtPYPDELFFRKNLLRLTG





2628
YQLsPTKLPSINFFRKNLLRLTG





2629
YQRPFSPsAYFFRKNLLRLTG





2630
YQYsDQGIDYFFRKNLLRLTG





2631
YRLsPEPTPLFFRKNLLRLTG





2632
YRPsYSYDYFFRKNLLRLTG





2633
YRPsYSYDYEFDFFRKNLLRLTG





2634
YRYDGQHFsLFFRKNLLRLTG





2635
YRYsLEKALFFRKNLLRLTG





2636
YSLDsPGPEKmALFFRKNLLRLTG





2637
YSLDsPGPEKMALFFRKNLLRLTG





2638
YSLsPSKSYKYFFRKNLLRLTG





2639
YSmsPGAMRFFRKNLLRLTG





2640
YSMsPGAmRFFRKNLLRLTG





2641
YSMsPGAMRFFRKNLLRLTG





2642
YVKLTPVsLFFRKNLLRLTG





2643
YVSsPDPQLFFRKNLLRLTG





2644
YYFsPSGKKFFFRKNLLRLTG





2645
yYISPRITFFFRKNLLRLTG





3997
DIAsLVGHEFFFRKNLLRLTG





3998
DIVsEYTHYFFRKNLLRLTG





3999
DSADLPPPsALFFRKNLLRLTG





4000
DVIDsQELSKVSREFFFRKNLLRLTG





4001
ETRSPsPISIFFRKNLLRLTG





4002
FKmIRSQsLFFRKNLLRLTG





4003
GAVsPGALRFFRKNLLRLTG





4004
GLPsPRGPGLFFRKNLLRLTG





4005
GRILsGVVTKFFRKNLLRLTG





4006
GRMIRAEsGPDLRYFFRKNLLRLTG





4007
GRmIRAEsGPDLRYFFRKNLLRLTG





4008
HPDGtPPKLFFRKNLLRLTG





4009
HPHLRKVsVFFRKNLLRLTG





4010
HRRIDIsPSTLFFRKNLLRLTG





4011
KAsSLISLLFFRKNLLRLTG





4012
KASsLISLLFFRKNLLRLTG





4013
KIPsAVSTVSMFFRKNLLRLTG





4014
KRFsMVVQDGIVKFFRKNLLRLTG





4015
KRFsmVVQDGIVKFFRKNLLRLTG





4016
KRFStEEFVLLFFRKNLLRLTG





4017
KRIsISISFFRKNLLRLTG





4018
KRIsISTSGFFRKNLLRLTG





4019
KRIsISTSGGFFRKNLLRLTG





4020
KRLsLDSSLVEYFFRKNLLRLTG





4021
KRLsLPADIRLFFRKNLLRLTG





4022
KRTsKYFSLFFRKNLLRLTG





4023
LPRsSSMAAGLFFRKNLLRLTG





4024
LPRSsSMAAGLFFRKNLLRLTG





4025
LQHsFSFAGFFFRKNLLRLTG





4026
LtSKLSTKDFFRKNLLRLTG





4027
NPTMLRTHsLFFRKNLLRLTG





4028
NRsSPVHIIFFRKNLLRLTG





4029
QVLPKtVKLFFFRKNLLRLTG





4030
RLPSPtSPFSSLFFRKNLLRLTG





4031
RPKLHHsLSFFFRKNLLRLTG





4032
RPRsDSLILFFRKNLLRLTG





4033
RQPswDPSPVFFRKNLLRLTG





4034
RRAsAPLPGLFFRKNLLRLTG





4035
RRASLsEIGFFRKNLLRLTG





4036
RRAsLSEIGFFRKNLLRLTG





4037
RRFsADEQFFFFRKNLLRLTG





4038
RRFsFSANFYFFRKNLLRLTG





4039
RRFsPPSSSLFFRKNLLRLTG





4040
RRIDIsPSFFRKNLLRLTG





4041
RRIsIVENcFFFRKNLLRLTG





4042
RRLPIFsRLSIFFRKNLLRLTG





4043
RRLsAIFLRLFFRKNLLRLTG





4044
RRLsFLVSYIFFRKNLLRLTG





4045
RRLsFTLERLFFRKNLLRLTG





4046
RRLsIEGNIAVFFRKNLLRLTG





4047
RRLsPPTLLFFRKNLLRLTG





4048
RSFSPtmKVFFRKNLLRLTG





4049
RSsSFTFHIFFRKNLLRLTG





4050
RSSsFTFHIFFRKNLLRLTG





4051
RtAATEVSLFFRKNLLRLTG





4052
RVDsTTCLFFFRKNLLRLTG





4053
RVDsTTcLFPFFRKNLLRLTG





4054
RVPsEHPYLFFRKNLLRLTG





4055
SAITPSVSRTsFFFRKNLLRLTG





4056
SEGsEPALLHFFRKNLLRLTG





4057
SIAsPDVKLNLFFRKNLLRLTG





4058
SIKsDVPVYFFRKNLLRLTG





4059
SLALtPPQAFFRKNLLRLTG





4060
SLKsRLRFFRKNLLRLTG





4061
SLPsPHPVRYFFRKNLLRLTG





4062
SPRPSPVPKPsPPLFFRKNLLRLTG





4063
SRFsSGGAFFRKNLLRLTG





4064
SRIVRTPsLFFRKNLLRLTG





4065
SRTSFTSVsRFFRKNLLRLTG





4066
TMPTsLPNLFFRKNLLRLTG





4067
TRLsPIAPAPGFFFRKNLLRLTG





4068
TSNsQKYmSFFFRKNLLRLTG





4069
TSTSRYLsLFFRKNLLRLTG





4070
VKTsGSSDRLFFRKNLLRLTG





4071
NIKsPALAFFFRKNLLRLTG





4072
LsPRAVSTTFFFRKNLLRLTG





4172
AHDPSGMFRSQsFFFRKNLLRLTG





4173
RVAsPAYSLFFRKNLLRLTG





4174
RRWtLGGMVNRFFRKNLLRLTG





4175
SIPSTLVsFFFRKNLLRLTG





4176
RRGsYPFIDFFFRKNLLRLTG





4177
LtLDQAYSYFFRKNLLRLTG





4178
SPPsPVEREmFFRKNLLRLTG





4179
SPPsPVEREMFFRKNLLRLTG





4180
LYVLsALLIFFRKNLLRLTG





4181
RPRsLSSPTVFFRKNLLRLTG





4182
LPIFNRIsVFFRKNLLRLTG





4183
IPRYHSQsPSmFFRKNLLRLTG





4184
SPLVRRPsLFFRKNLLRLTG





4185
EAPKVSRsLFFRKNLLRLTG





4186
SLDSPsYVLYFFRKNLLRLTG





4187
REYsPPYAPFFRKNLLRLTG





4188
YGYEGSEsIFFRKNLLRLTG





4189
RPSsLPLDFFFRKNLLRLTG





4190
RPsSLPLDFFFRKNLLRLTG





4191
TPItPLKDGFFFRKNLLRLTG





4192
KRFsFKKSFKLFFRKNLLRLTG





4193
KRNsRLGFLYFFRKNLLRLTG





4194
RRAsAILPGVLFFRKNLLRLTG





‘s’, ‘t’, and ‘y’ stand for phosphoserine, phosphothreonine, and phosphotyrosine, respectively.


‘m’ stands for oxidized methionine.


‘w’ stands for oxidized tryptophan.


‘c’ stands for cysteinylated cysteine.













TABLE 4







Amino acid sequences of exemplary


antigenic polypeptides








SEQ



ID



NO
Amino Acid Sequence





2646
AELGRLsPRAYFFRKNWLRLTW





2647
AESImsFHIFFRKNWLRLTW





2648
AESIMsFHIFFRKNWLRLTW





2649
AEsLKSLSSELFFRKNWLRLTW





2650
AEtPDIKLFFFRKNWLRLTW





2651
AGFsFVNPKFFRKNWLRLTW





2652
AHDPSGmFRSQsFFFRKNWLRLTW





2653
ALDSGAsLLHLFFRKNWLRLTW





2654
ALmGsPQLVAAFFRKNWLRLTW





2655
ALPPGSYAsLFFRKNWLRLTW





2656
ALPTPALsPSLMFFRKNWLRLTW





2657
ALSsSFLVLFFRKNWLRLTW





2658
ALSSsFLVLFFRKNWLRLTW





2659
ALStPVVEKFFRKNWLRLTW





2660
ALVDGyFRLFFRKNWLRLTW





2661
ALwsPGLAKFFRKNWLRLTW





2662
AmLGSKsPDPYRLFFRKNWLRLTW





2663
APAsPFRQLFFRKNWLRLTW





2664
APAsPLRPLFFRKNWLRLTW





2665
APAsPNHAGVLFFRKNWLRLTW





2666
APFHLtPTLYFFRKNWLRLTW





2667
APKsPSSEWLFFRKNWLRLTW





2668
APRtPPGVTFFFRKNWLRLTW





2669
APsSPDVKLFFRKNWLRLTW





2670
APSsPDVKLFFRKNWLRLTW





2671
APTsPLGHLFFRKNWLRLTW





2672
APVsPRPGLFFRKNWLRLTW





2673
ARFsGFYSmFFRKNWLRLTW





2674
ARFsGFYSMFFRKNWLRLTW





2675
ARFsPKVSLFFRKNWLRLTW





2676
ARGIsPIVFFFRKNWLRLTW





2677
ARYsGSYNDYFFRKNWLRLTW





2678
ASFKAELsYFFRKNWLRLTW





2679
ASFtPTSILKFFRKNWLRLTW





2680
ASFtPTSILKRFFRKNWLRLTW





2681
ASLsPSVSKFFRKNWLRLTW





2682
ATIsPPLQPKFFRKNWLRLTW





2683
AVILPPLsPYFKFFRKNWLRLTW





2684
AVLEyLKIFFRKNWLRLTW





2685
AVNQFsPSLARFFRKNWLRLTW





2686
AVRNFsPTDYYFFRKNWLRLTW





2687
AVRNFSPtDYYFFRKNWLRLTW





2688
AWRRLsRDSGGYFFRKNWLRLTW





2689
AYGGLtSPGLSYFFRKNWLRLTW





2690
AYGGLTsPGLSYFFRKNWLRLTW





2691
AYSsYVHQYFFRKNWLRLTW





2692
CtFGSRQIFFRKNWLRLTW





2693
DFAsPFHERFFRKNWLRLTW





2694
DFHsPIVLGRFFRKNWLRLTW





2695
DIAsPTFRRLFFRKNWLRLTW





2696
DIIRQPsEEEIIKFFRKNWLRLTW





2697
DIKsVFEAFFFRKNWLRLTW





2698
DILsPRLIRFFRKNWLRLTW





2699
DIRRFsLTTLRFFRKNWLRLTW





2700
DIsPPIFRRFFRKNWLRLTW





2701
DLtLKKEKFFFRKNWLRLTW





2702
DMLGLtKPAMPMFFRKNWLRLTW





2703
DNFsPDLRVLRFFRKNWLRLTW





2704
DPFGRPTsFFFRKNWLRLTW





2705
DPLIRWDsYFFRKNWLRLTW





2706
DPSLDLHsLFFRKNWLRLTW





2707
DSDPmLsPRFYFFRKNWLRLTW





2708
DSDPMLsPRFYFFRKNWLRLTW





2709
DSDPmLsPRFYAYFFRKNWLRLTW





2710
DSDPMLsPRFYAYFFRKNWLRLTW





2711
DsGEGDFLAEGGGVRFFRKNWLRLTW





2712
DSKsPLGFYFFRKNWLRLTW





2713
DTIsLASERYFFRKNWLRLTW





2714
DTIsPTLGFFFRKNWLRLTW





2715
DTQSGsLLFIGRFFRKNWLRLTW





2716
DTsSLPTVIMRFFRKNWLRLTW





2717
DTSsLPTVImRFFRKNWLRLTW





2718
DTSsLPTVIMRFFRKNWLRLTW





2719
DTTsLRTLRIFFRKNWLRLTW





2720
DVAsPDGLGRLFFRKNWLRLTW





2721
DVAsPTLRFFRKNWLRLTW





2722
DVAsPTLRRFFRKNWLRLTW





2723
DVAsPTLRRLFFRKNWLRLTW





2724
DVIDsQELSKVFFRKNWLRLTW





2725
DVYSGtPTKVFFRKNWLRLTW





2726
DYSPYFKtIFFRKNWLRLTW





2727
EAsSPVPYLFFRKNWLRLTW





2728
EASsPVPYLFFRKNWLRLTW





2729
EEAPQtPVAFFFRKNWLRLTW





2730
EEDtYEKVFFFRKNWLRLTW





2731
EEFsPRQAQmFFFRKNWLRLTW





2732
EEFsPRQAQMFFFRKNWLRLTW





2733
EEIsPTKFPGLFFRKNWLRLTW





2734
EEIsPTKFPGLYFFRKNWLRLTW





2735
EELsPLALGRFFFRKNWLRLTW





2736
EELsPSTVLYFFRKNWLRLTW





2737
EELSPsTVLYFFRKNWLRLTW





2738
EELSPtAKFFFRKNWLRLTW





2739
EGPEtGYSLFFRKNWLRLTW





2740
EHERSIsPLLFFFRKNWLRLTW





2741
EIVNFsPIARFFRKNWLRLTW





2742
ERLKIRGsLFFRKNWLRLTW





2743
ERVDSLVsLFFRKNWLRLTW





2744
ESFSDyPPLGRFAFFRKNWLRLTW





2745
ESLsPIGDmKVFFRKNWLRLTW





2746
ESLsPIGDMKVFFRKNWLRLTW





2747
ESVYKASLsLFFRKNWLRLTW





2748
ETRRPsYLEWFFRKNWLRLTW





2749
EVIRKGsITEYFFRKNWLRLTW





2750
EVIsQHLVSYFFRKNWLRLTW





2751
EVIsVLQKYFFRKNWLRLTW





2752
EVLERKIsMFFRKNWLRLTW





2753
FAFPGStNSLFFRKNWLRLTW





2754
FAFPGSTNsLFFRKNWLRLTW





2755
FASPtSPPVLFFRKNWLRLTW





2756
FASPTsPPVLFFRKNWLRLTW





2757
FATIKSAsLFFRKNWLRLTW





2758
FATIRTAsLFFRKNWLRLTW





2759
FAVsPIPGRGGVLFFRKNWLRLTW





2760
FAwsPLAGEKFFFRKNWLRLTW





2761
FAWsPLAGEKFFFRKNWLRLTW





2762
FAYsPGGAHGmLFFRKNWLRLTW





2763
FFFtARTSFFFRKNWLRLTW





2764
FGGQRLtLFFRKNWLRLTW





2765
FHGISTVsLFFRKNWLRLTW





2766
FHVtPLKLFFRKNWLRLTW





2767
FIVsPVPESRLFFRKNWLRLTW





2768
FKVsPLTFGRFFRKNWLRLTW





2769
FLDsAYFRLFFRKNWLRLTW





2770
FLDsGTIRGVFFRKNWLRLTW





2771
FLFsPPEVTGRFFRKNWLRLTW





2772
FLKPsTSGDSLFFRKNWLRLTW





2773
FLKPSTsGDSLFFRKNWLRLTW





2774
FLKPSTSGDsLFFRKNWLRLTW





2775
FLNEKARLsYFFRKNWLRLTW





2776
FLsRSIPSLFFRKNWLRLTW





2777
FPDNsDVSSIGRLFFRKNWLRLTW





2778
FPDNSDVSsIGRLFFRKNWLRLTW





2779
FPLMRSKsLFFRKNWLRLTW





2780
FPLsPTKLSQYFFRKNWLRLTW





2781
FPSMPsPRLFFRKNWLRLTW





2782
FQYSKSPsLFFRKNWLRLTW





2783
FRFsPMGVDHMFFRKNWLRLTW





2784
FRPPPLtPEDVGFFFRKNWLRLTW





2785
FRRPDIQYPDAtDEFFRKNWLRLTW





2786
FRRsDDMFTFFFRKNWLRLTW





2787
FRYSGKtEYFFRKNWLRLTW





2788
FSFKKsFKLFFRKNWLRLTW





2789
FSFsPGAGAFRFFRKNWLRLTW





2790
FSLRYsPGmDAYFFRKNWLRLTW





2791
FSLRYsPGMDAYFFRKNWLRLTW





2792
FSRPSMsPTPLDRFFRKNWLRLTW





2793
FSVDsPRIYFFRKNWLRLTW





2794
FTIFRTIsVFFRKNWLRLTW





2795
FtPPVVKRFFRKNWLRLTW





2796
FVLsPIKEPAFFRKNWLRLTW





2797
FVRsPGTGAFFFRKNWLRLTW





2798
FVtTPTAELFFRKNWLRLTW





2799
FVTtPTAELFFRKNWLRLTW





2800
FVTTPtAELFFRKNWLRLTW





2801
FYYsPSGKKFFFRKNWLRLTW





2802
GALsRYLFRFFRKNWLRLTW





2803
GEDPLsPRALFFRKNWLRLTW





2804
GELEsIGELFFFRKNWLRLTW





2805
GEmsPQRFFFFRKNWLRLTW





2806
GEMsPQRFFFFRKNWLRLTW





2807
GEmsPQRFFFFFRKNWLRLTW





2808
GENKsPLLLFFRKNWLRLTW





2809
GEPRAPtPPSGTEVTLFFRKNWLRLT



W





2810
GEPsPPHDILFFRKNWLRLTW





2811
GEtSPRTKITWFFRKNWLRLTW





2812
GETsPRTKITWFFRKNWLRLTW





2813
GEwsASLPHRFFFRKNWLRLTW





2814
GEwSAsLPHRFFFRKNWLRLTW





2815
GEWsASLPHRFFFRKNWLRLTW





2816
GEYsPGTALPFFRKNWLRLTW





2817
GGLTsPGLSYFFRKNWLRLTW





2818
GGSISVQVNSIKFDsEFFRKNWLRLT



W





2819
GHGsPFPSLFFRKNWLRLTW





2820
GIFPGtPLKKFFRKNWLRLTW





2821
GIISsPLTGKFFRKNWLRLTW





2822
GIISSPLtGKFFRKNWLRLTW





2823
GImsPLAKKFFRKNWLRLTW





2824
GLFsPIRSSAFFFRKNWLRLTW





2825
GLLsLSALGSQAHLFFRKNWLRLTW





2826
GLPGGGsPTTFLFFRKNWLRLTW





2827
GLSsLSIHLFFRKNWLRLTW





2828
GLTsPGLSYSLFFRKNWLRLTW





2829
GLtVSIPGLFFRKNWLRLTW





2830
GMATLsLLLKFFRKNWLRLTW





2831
GPGHHHKPGLGEGtPFFRKNWLRLTW





2832
GPLSRVKsLFFRKNWLRLTW





2833
GPLVRQIsLFFRKNWLRLTW





2834
GPRAPSPtKPLFFRKNWLRLTW





2835
GPRsASLLFFRKNWLRLTW





2836
GPRSFtPLSIFFRKNWLRLTW





2837
GPRsPKAWLFFRKNWLRLTW





2838
GPRtPTQPLLFFRKNWLRLTW





2839
GRNsLSSLPTYFFRKNWLRLTW





2840
GRQSPsFKLFFRKNWLRLTW





2841
GSFAsPGRLFFFRKNWLRLTW





2842
GsFRGFPALFFRKNWLRLTW





2843
GSKsPDPYRLFFRKNWLRLTW





2844
GSRsLYNLRFFRKNWLRLTW





2845
GTFPKALsIFFRKNWLRLTW





2846
GtPLSQATIHQYFFRKNWLRLTW





2847
GTVtPPPRLVKFFRKNWLRLTW





2848
GTYVPSsPTRLAYFFRKNWLRLTW





2849
GVIKsPSWQRFFRKNWLRLTW





2850
GVIsPQELLKFFRKNWLRLTW





2851
GVIsPQELLKKFFRKNWLRLTW





2852
GVLsPDTISSKFFRKNWLRLTW





2853
GVmtPLIKRFFRKNWLRLTW





2854
GVMtPLIKRFFRKNWLRLTW





2855
HEFsSPSHLLFFRKNWLRLTW





2856
HEFSsPSHLLFFRKNWLRLTW





2857
HELsDITELFFRKNWLRLTW





2858
HERSIsPLLFFRKNWLRLTW





2859
HFDsPPHLLFFRKNWLRLTW





2860
HHHKPGLGEGtPFFRKNWLRLTW





2861
HHPGLGEGtPFFRKNWLRLTW





2862
HKIsDYFEYFFRKNWLRLTW





2863
HLLEtTPKSEFFRKNWLRLTW





2864
HLLETtPKSEFFRKNWLRLTW





2865
HLLSPtKGIFFRKNWLRLTW





2866
HLNsLDVQLFFRKNWLRLTW





2867
HLPsPPLTQEVFFRKNWLRLTW





2868
HLSsFTMKLFFRKNWLRLTW





2869
HPIsPYEHLFFRKNWLRLTW





2870
HPIsPYEHLLFFRKNWLRLTW





2871
HPIsSEELLFFRKNWLRLTW





2872
HPISsEELLFFRKNWLRLTW





2873
HPIsSEELLSLKYFFRKNWLRLTW





2874
HPISsEELLSLKYFFRKNWLRLTW





2875
HPRPVPDsPVSVTRLFFRKNWLRLTW





2876
HPRsPNVLSVALFFRKNWLRLTW





2877
HPsLSAPALFFRKNWLRLTW





2878
HPSLsAPALFFRKNWLRLTW





2879
HPTLQAPsLFFRKNWLRLTW





2880
HPYRNsDPVIFFRKNWLRLTW





2881
HQFsLKENwFFRKNWLRLTW





2882
HQGKFLQtFFFRKNWLRLTW





2883
HRAsKVLFLFFRKNWLRLTW





2884
HRDsFSRmSLFFRKNWLRLTW





2885
HRDsFSRMSLFFRKNWLRLTW





2886
HRNsmKVFLFFRKNWLRLTW





2887
HRVsVILKLFFRKNWLRLTW





2888
HSDKRRPPsAELYFFRKNWLRLTW





2889
HSLsLDDIRLYFFRKNWLRLTW





2890
HSVsPDPVLFFRKNWLRLTW





2891
HTIsPLDLAFFRKNWLRLTW





2892
HTIsPLDLAKFFRKNWLRLTW





2893
HTIsPLDLAKLFFRKNWLRLTW





2894
HTIsPSFQLFFRKNWLRLTW





2895
HTISPsFQLFFRKNWLRLTW





2896
HVSLITPtKRFFRKNWLRLTW





2897
HYFsPFRPYFFRKNWLRLTW





2898
HYsSRLGSAIFFFRKNWLRLTW





2899
HYSsRLGSAIFFFRKNWLRLTW





2900
HYSSRLGsAIFFFRKNWLRLTW





2901
IAATKsLSVFFRKNWLRLTW





2902
IEIERILsVFFRKNWLRLTW





2903
IFDLQKTsLFFRKNWLRLTW





2904
IIQsPSSTGLLKFFRKNWLRLTW





2905
ILGPPPPsFHLFFRKNWLRLTW





2906
ILLtDLIIFFRKNWLRLTW





2907
IMKNLQAHyEFFRKNWLRLTW





2908
IPHQRSsLFFRKNWLRLTW





2909
IPKsKFLALFFRKNWLRLTW





2910
IPMtPTSSFFFRKNWLRLTW





2911
IPMTPtSSFFFRKNWLRLTW





2912
IPRPLsLIGFFRKNWLRLTW





2913
IPRsFRHLSFFFRKNWLRLTW





2914
IPsmSHVHLFFRKNWLRLTW





2915
IPsMSHVHLFFRKNWLRLTW





2916
IPsPLQPEmFFRKNWLRLTW





2917
IPsPLQPEMFFRKNWLRLTW





2918
IPVSKPLsLFFRKNWLRLTW





2919
IPVsRDWELFFRKNWLRLTW





2920
IRFGRKPsLFFRKNWLRLTW





2921
IRPsVLGPLFFRKNWLRLTW





2922
IRRsYFEVFFFRKNWLRLTW





2923
IRYSGHsLFFRKNWLRLTW





2924
ISKKLsFLSWFFRKNWLRLTW





2925
ISLDKLVsIFFRKNWLRLTW





2926
IsSLTTLSIFFRKNWLRLTW





2927
ISsLTTLSIFFRKNWLRLTW





2928
IssSmHSLYFFRKNWLRLTW





2929
ISsSMHSLYFFRKNWLRLTW





2930
ISSsmHSLYFFRKNWLRLTW





2931
ITItPPEKYFFRKNWLRLTW





2932
ITLLsPKHKYFFRKNWLRLTW





2933
ItPPSSEKLVSVmFFRKNWLRLTW





2934
ItPPSSEKLVSVMFFRKNWLRLTW





2935
ITTsPITVRFFRKNWLRLTW





2936
ITTsPITVRKFFRKNWLRLTW





2937
ITYsPKLERFFRKNWLRLTW





2938
IVLPLsLQRFFRKNWLRLTW





2939
IVsSLRLAYFFRKNWLRLTW





2940
IVSsLRLAYFFRKNWLRLTW





2941
IYDsVKVYFFFRKNWLRLTW





2942
IYRSQsPHYFFFRKNWLRLTW





2943
KAFsESGSNLHALFFRKNWLRLTW





2944
KAFsPVRSVRFFRKNWLRLTW





2945
KAFsPVRSVRKFFRKNWLRLTW





2946
KAItPPQQPYFFRKNWLRLTW





2947
KASsPGHPAFFFRKNWLRLTW





2948
KAVsFHLVHFFRKNWLRLTW





2949
KAVsLFLFFRKNWLRLTW





2950
KAYtPVVVTQWFFRKNWLRLTW





2951
KEDsFLQRYFFRKNWLRLTW





2952
KEmSPtRQLFFRKNWLRLTW





2953
KEsEVFYELFFRKNWLRLTW





2954
KEsTLHLVLFFRKNWLRLTW





2955
KEStLHLVLFFRKNWLRLTW





2956
KFLsPAQYLYFFRKNWLRLTW





2957
KFRDLsPPRYFFRKNWLRLTW





2958
KFsLRAAEFFFRKNWLRLTW





2959
KGFsGTFQLFFRKNWLRLTW





2960
KIFERATsFFFRKNWLRLTW





2961
KIFsKQQGKAFQRFFRKNWLRLTW





2962
KIIsIFSGFFRKNWLRLTW





2963
KIIsIFSGTEKFFRKNWLRLTW





2964
KIKsLEEIYLFFRKNWLRLTW





2965
KINsLAHLRFFRKNWLRLTW





2966
KISsFTSLKFFRKNWLRLTW





2967
KISSFtSLKFFRKNWLRLTW





2968
KISSFTsLKFFRKNWLRLTW





2969
KISsLEIKLFFRKNWLRLTW





2970
KKLsLLNGGLFFRKNWLRLTW





2971
KLEGPDVsLFFRKNWLRLTW





2972
KLFHGsLEELFFRKNWLRLTW





2973
KLFPGsPATYFFRKNWLRLTW





2974
KLHsLIGLGIFFRKNWLRLTW





2975
KLIDIVSsQKVFFRKNWLRLTW





2976
KLKsFTYEYFFRKNWLRLTW





2977
KLLDFGsLSNLFFRKNWLRLTW





2978
KLLEGEESRIsLFFRKNWLRLTW





2979
KLLsPILARYFFRKNWLRLTW





2980
KLLsTALHVFFRKNWLRLTW





2981
KLLsYIQRLFFRKNWLRLTW





2982
KLMsDVEDVSLFFRKNWLRLTW





2983
KLMsLGDIRLFFRKNWLRLTW





2984
KLmsPKADVKLFFRKNWLRLTW





2985
KLMsPVLKQHLFFRKNWLRLTW





2986
KLQEFsKEEFFRKNWLRLTW





2987
KLRIQtDGDKYFFRKNWLRLTW





2988
KLSsGLLPKLFFRKNWLRLTW





2989
KLwtLVSEQTRVFFRKNWLRLTW





2990
KLWtLVSEQTRVFFRKNWLRLTW





2991
KLYRPGsVAYFFRKNWLRLTW





2992
KLYsISSQVFFRKNWLRLTW





2993
KLYsPTSKALFFRKNWLRLTW





2994
KLYSPtSKALFFRKNWLRLTW





2995
KLYTyIQSRFFRKNWLRLTW





2996
KLYTyIQSRFFFRKNWLRLTW





2997
KmDsFLDMQLFFRKNWLRLTW





2998
KMDsFLDmQLFFRKNWLRLTW





2999
KmsSYAFFVFFRKNWLRLTW





3000
KmSsYAFFVFFRKNWLRLTW





3001
KMsSYAFFVFFRKNWLRLTW





3002
KMSsYAFFVFFRKNWLRLTW





3003
KmsSYAFFVQTFFRKNWLRLTW





3004
KmSsYAFFVQTFFRKNWLRLTW





3005
KMsSYAFFVQTFFRKNWLRLTW





3006
KMSsYAFFVQTFFRKNWLRLTW





3007
KPAsPARRLDLFFRKNWLRLTW





3008
KPDKTLRFsLFFRKNWLRLTW





3009
KPHsPVTGLYLFFRKNWLRLTW





3010
KPLsRVTSLFFRKNWLRLTW





3011
KPPsPGTVLFFRKNWLRLTW





3012
KPPSPGtVLFFRKNWLRLTW





3013
KPRPLsmDLFFRKNWLRLTW





3014
KPRSIsFPSAFFRKNWLRLTW





3015
KPSSLRRVtIFFRKNWLRLTW





3016
KPSsPRGSLLLFFRKNWLRLTW





3017
KQKsLTNLSFFFRKNWLRLTW





3018
KQKSLtNLSFFFRKNWLRLTW





3019
KRAsALLNLFFRKNWLRLTW





3020
KRAsYELEFFFRKNWLRLTW





3021
KRDsFIGTPYFFRKNWLRLTW





3022
KRFsLDFNLFFRKNWLRLTW





3023
KRIsIFLSMFFRKNWLRLTW





3024
KRIsISTSGGSFFFRKNWLRLTW





3025
KRLGsLVDEFFFRKNWLRLTW





3026
KRLsVELTSSLFFRKNWLRLTW





3027
KRLsVELTSSLFFFRKNWLRLTW





3028
KRLsVERIYQKFFRKNWLRLTW





3029
KRMsFVMEYFFRKNWLRLTW





3030
KRNsDLLLLFFRKNWLRLTW





3031
KRPsSEDFVFFFRKNWLRLTW





3032
KRPsSEDFVFLFFRKNWLRLTW





3033
KRPSsEDFVFLFFRKNWLRLTW





3034
KRRtGALVLFFRKNWLRLTW





3035
KRSsISQLLFFRKNWLRLTW





3036
KRVsTFQEFFFRKNWLRLTW





3037
KRVtWIVEFFFRKNWLRLTW





3038
KRYLFRsFFFRKNWLRLTW





3039
KRYsRSLTIFFRKNWLRLTW





3040
KSAsFAFEFFFRKNWLRLTW





3041
KSDGsFIGYFFRKNWLRLTW





3042
KSFsAPATQAYFFRKNWLRLTW





3043
KSGELLAtwFFRKNWLRLTW





3044
KSGEPLStWFFRKNWLRLTW





3045
KSKsIEITFFFRKNWLRLTW





3046
KsLPSDQVmLFFRKNWLRLTW





3047
KsLPSDQVMLFFRKNWLRLTW





3048
KSLsIEIGHEVFFRKNWLRLTW





3049
KSLSPsLLGYFFRKNWLRLTW





3050
KSSEEKRLSIsKFFFRKNWLRLTW





3051
KSSsLPRAFFFRKNWLRLTW





3052
KSVtPTKEFLFFRKNWLRLTW





3053
KTDsDSDLQLYFFRKNWLRLTW





3054
KTIsESDLNHSFFFRKNWLRLTW





3055
KTIsPKSTVYFFRKNWLRLTW





3056
KTKsMFFFLFFRKNWLRLTW





3057
KTLsLVKELFFRKNWLRLTW





3058
KTmsGTFLLFFRKNWLRLTW





3059
KTmSGtFLLFFRKNWLRLTW





3060
KTMSGtFLLFFRKNWLRLTW





3061
KTmsGTFLLRFFFRKNWLRLTW





3062
KTMsGTFLLRFFFRKNWLRLTW





3063
KtMSPSQMIMFFRKNWLRLTW





3064
KTQRVsLLFFFRKNWLRLTW





3065
KtRSLSVEIVYFFRKNWLRLTW





3066
KTRsLSVEIVYFFRKNWLRLTW





3067
KTVsPPIRKGWFFRKNWLRLTW





3068
KTVsSTKLVSFFFRKNWLRLTW





3069
KVDGPRSPsYFFRKNWLRLTW





3070
KVEsPPLEEwFFRKNWLRLTW





3071
KVFsLPTQLFFRKNWLRLTW





3072
KVFsPVIRSSFFFRKNWLRLTW





3073
KVGsFKFIYVFFRKNWLRLTW





3074
KVLswPFLmFFRKNWLRLTW





3075
KVLswPFLMFFRKNWLRLTW





3076
KWPsKRRIPVFFRKNWLRLTW





3077
KYRsVISDIFFFRKNWLRLTW





3078
LAFPsPEKLLRFFRKNWLRLTW





3079
LAsDRCSIHLFFRKNWLRLTW





3080
LEIKEsILSLFFRKNWLRLTW





3081
LEIsPDNSLFFRKNWLRLTW





3082
LEIsVGKSVFFRKNWLRLTW





3083
LEsPTTPLLFFRKNWLRLTW





3084
LESPtTPLLFFRKNWLRLTW





3085
LESPTtPLLFFRKNWLRLTW





3086
LGFEVKsKmVFFRKNWLRLTW





3087
LGFEVKsKMVFFRKNWLRLTW





3088
LGmEVLsGVFFRKNWLRLTW





3089
LGMEVLsGVFFRKNWLRLTW





3090
LIPDHtIRAFFRKNWLRLTW





3091
LLDIIRsLFFRKNWLRLTW





3092
LLDPRSYHtYFFRKNWLRLTW





3093
LLsPKHKYFFRKNWLRLTW





3094
LPAsPRARLSAFFRKNWLRLTW





3095
LPAsPSVSLFFRKNWLRLTW





3096
LPASPsVSLFFRKNWLRLTW





3097
LPDPGsPRLFFRKNWLRLTW





3098
LPEsPRLTLFFRKNWLRLTW





3099
LPFSGPREPsLFFRKNWLRLTW





3100
LPFSsSPSRSAFFRKNWLRLTW





3101
LPFSSsPSRSAFFRKNWLRLTW





3102
LPLsSSHLNVYFFRKNWLRLTW





3103
LPLSsSHLNVYFFRKNWLRLTW





3104
LPLSSsHLNVYFFRKNWLRLTW





3105
LPPVsPLKAAFFRKNWLRLTW





3106
LPRGLsPARQLFFRKNWLRLTW





3107
LPRGSSPsVLFFRKNWLRLTW





3108
LPRPLsPTKLFFRKNWLRLTW





3109
LPRPLSPtKLFFRKNWLRLTW





3110
LPRRLsDSPVFFFRKNWLRLTW





3111
LPRRLSDsPVFFFRKNWLRLTW





3112
LPRsPPLKVLFFRKNWLRLTW





3113
LPRsSRGLLFFRKNWLRLTW





3114
LPRSsRGLLFFRKNWLRLTW





3115
LPRSSsmAAGLFFRKNWLRLTW





3116
LPSARPLsLFFRKNWLRLTW





3117
LPsRLTKcFFRKNWLRLTW





3118
LPTsPLAmFFRKNWLRLTW





3119
LPtSPLAmEYFFRKNWLRLTW





3120
LPtSPLAMEYFFRKNWLRLTW





3121
LPTsPLAmEYFFRKNWLRLTW





3122
LPTsPLAMEYFFRKNWLRLTW





3123
LPVsPGHRKTFFRKNWLRLTW





3124
LPYPVsPKQKYFFRKNWLRLTW





3125
LQHSFsFAGFFFRKNWLRLTW





3126
LQIsPVSSYFFRKNWLRLTW





3127
LSKsSATLwFFRKNWLRLTW





3128
LSPtKLPSIFFRKNWLRLTW





3129
LSRTFKsLFFFRKNWLRLTW





3130
LsSSVIRELFFRKNWLRLTW





3131
LSsSVIRELFFRKNWLRLTW





3132
LTAsQILSRFFRKNWLRLTW





3133
LTDPsSPTISSYFFRKNWLRLTW





3134
LTDPSSPtISSYFFRKNWLRLTW





3135
LTKtLIKLFFRKNWLRLTW





3136
LVAsPRLEKFFRKNWLRLTW





3137
LVREPGsQAcLFFRKNWLRLTW





3138
mIIsPERLDPFFFRKNWLRLTW





3139
MIIsPERLDPFFFRKNWLRLTW





3140
MLPsPNEKLFFRKNWLRLTW





3141
MPFPAHLtYFFRKNWLRLTW





3142
mPHsPTLRVFFRKNWLRLTW





3143
mPHSPtLRVFFRKNWLRLTW





3144
MPHsPTLRVFFRKNWLRLTW





3145
MPHSPtLRVFFRKNWLRLTW





3146
MPKFRMPsLFFRKNWLRLTW





3147
MPQDLRsPAFFRKNWLRLTW





3148
mPREPsATRLFFRKNWLRLTW





3149
mPRQPsATRLFFRKNWLRLTW





3150
mPsPATLSHSLFFRKNWLRLTW





3151
MPsPATLSHSLFFRKNWLRLTW





3152
MPsPFRSSALFFRKNWLRLTW





3153
mPsPGGRITLFFRKNWLRLTW





3154
MPsPGGRITLFFRKNWLRLTW





3155
MPsPIMHPLILFFRKNWLRLTW





3156
MPsPLKGQHTLFFRKNWLRLTW





3157
MPsPSTLKKELFFRKNWLRLTW





3158
mPsPVSPKLFFRKNWLRLTW





3159
mPSPVsPKLFFRKNWLRLTW





3160
MPsPVSPKLFFRKNWLRLTW





3161
MPSPVsPKLFFRKNWLRLTW





3162
MPtSPGVDLFFRKNWLRLTW





3163
MPTsPGVDLFFRKNWLRLTW





3164
mRLsRELQLFFRKNWLRLTW





3165
MSKLINHtFFRKNWLRLTW





3166
mTKSsPLKIFFRKNWLRLTW





3167
NAIsLPTIFFRKNWLRLTW





3168
NAVsPSSGPSLFFRKNWLRLTW





3169
NAWsPVMRARFFRKNWLRLTW





3170
NHVtPPNVSLFFRKNWLRLTW





3171
NIPsFIVRLFFRKNWLRLTW





3172
NLLsPDGKmISVFFRKNWLRLTW





3173
NmDsPGPMLFFRKNWLRLTW





3174
NMDsPGPmLFFRKNWLRLTW





3175
NPIHsPSYPLFFRKNWLRLTW





3176
NPIHSPsYPLFFRKNWLRLTW





3177
NPsSPEFFmFFRKNWLRLTW





3178
NPsSPEFFMFFRKNWLRLTW





3179
NPSsPEFFmFFRKNWLRLTW





3180
NPSsPEFFMFFRKNWLRLTW





3181
NQGsPFKSALFFRKNWLRLTW





3182
NREsFQIFLFFRKNWLRLTW





3183
NRFsGGFGARDYFFRKNWLRLTW





3184
NRFsPKASLFFRKNWLRLTW





3185
NRHsLPFSLFFRKNWLRLTW





3186
NRHsLVEKLFFRKNWLRLTW





3187
NRLsLLVQKFFRKNWLRLTW





3188
NRMsRRIVLFFRKNWLRLTW





3189
NRSLHINNIsPGNTISFFRKNWLRLT



W





3190
NRSsPVHIIFFRKNWLRLTW





3191
NSISSVVsRFFRKNWLRLTW





3192
NSLsPRSSLFFRKNWLRLTW





3193
NSVsPSESLFFRKNWLRLTW





3194
NVLsPLPSQFFRKNWLRLTW





3195
NVLsPLPSQAMFFRKNWLRLTW





3196
NVMKRKFsLFFRKNWLRLTW





3197
PEFPLsPPKKFFRKNWLRLTW





3198
PEVsPRPALFFRKNWLRLTW





3199
PIFSRLsIFFRKNWLRLTW





3200
PVSKPLsLFFRKNWLRLTW





3201
QEAsPRPLLFFRKNWLRLTW





3202
QLMtLENKLFFRKNWLRLTW





3203
QLPsPTATSQLFFRKNWLRLTW





3204
QPRNSLPAsPAHQLFFRKNWLRLTW





3205
QPRTPsPLVLFFRKNWLRLTW





3206
QRVPsYDSFFFRKNWLRLTW





3207
QSIsFSGLPSGRFFRKNWLRLTW





3208
QSSsWTRVFFFRKNWLRLTW





3209
QTIsPLSTYFFRKNWLRLTW





3210
QTPDFtPTKYFFRKNWLRLTW





3211
QTPsPRLALFFRKNWLRLTW





3212
QTRRPsYLEWFFRKNWLRLTW





3213
RAAsIENVLFFRKNWLRLTW





3214
RAAsSPDGFFwFFRKNWLRLTW





3215
RASsPDGFFwFFRKNWLRLTW





3216
RAAtPLPSLFFRKNWLRLTW





3217
RAAtPTLTTFFFRKNWLRLTW





3218
RAATPtLTTFFFRKNWLRLTW





3219
RAGsFSRFYFFRKNWLRLTW





3220
RAHtPTPGIYmFFRKNWLRLTW





3221
RAHtPTPGIYMFFRKNWLRLTW





3222
RAHTPtPGIYMFFRKNWLRLTW





3223
RALsHADLFFFRKNWLRLTW





3224
RALsLTRALFFRKNWLRLTW





3225
RANsFVGTAQYFFRKNWLRLTW





3226
RAPsYRTLELFFRKNWLRLTW





3227
RARsPVLWGWFFRKNWLRLTW





3228
RAsSLNFLNKFFRKNWLRLTW





3229
RASsLNFLNKFFRKNWLRLTW





3230
RAtSNVFAmFFRKNWLRLTW





3231
RAtSNVFAMFFRKNWLRLTW





3232
RATsNVFAmFFRKNWLRLTW





3233
RATsNVFAMFFRKNWLRLTW





3234
RAtSNVFAmFFFRKNWLRLTW





3235
RAtSNVFAMFFFRKNWLRLTW





3236
RATsNVFAmFFFRKNWLRLTW





3237
RATsNVFAMFFFRKNWLRLTW





3238
RATsPLVSLYFFRKNWLRLTW





3239
RAVsPFAKIFFRKNWLRLTW





3240
RAVsPHFDDmFFRKNWLRLTW





3241
RAVsPHFDDMFFRKNWLRLTW





3242
RAYsPLHGGSGSYFFRKNWLRLTW





3243
REAPsPLmFFRKNWLRLTW





3244
REAPsPLMFFRKNWLRLTW





3245
REAsIELPSmFFRKNWLRLTW





3246
REDsLEFSLFFRKNWLRLTW





3247
REDSLEFsLFFRKNWLRLTW





3248
REFSGPStPTGTLFFRKNWLRLTW





3249
REFSGPSTPtGTLFFRKNWLRLTW





3250
REImGtPEYLFFRKNWLRLTW





3251
RELsAPARLYFFRKNWLRLTW





3252
RELsGTIKEILFFRKNWLRLTW





3253
RELsPSSLKmFFRKNWLRLTW





3254
RELsPVSFQYFFRKNWLRLTW





3255
REPsESSPLALFFRKNWLRLTW





3256
REPSESsPLALFFRKNWLRLTW





3257
REPsPLPELALFFRKNWLRLTW





3258
REPsPVRYDNLFFRKNWLRLTW





3259
RERAFsVKFFFRKNWLRLTW





3260
REsPIPIEIFFRKNWLRLTW





3261
REsPRPLQLFFRKNWLRLTW





3262
RESsLGFQLFFRKNWLRLTW





3263
RETNLDsLPLFFRKNWLRLTW





3264
RETsMVHELFFRKNWLRLTW





3265
RETsPNRIGLFFRKNWLRLTW





3266
REVsPEPIVFFRKNWLRLTW





3267
RFQsmPVRLFFRKNWLRLTW





3268
RFQsMPVRLFFRKNWLRLTW





3269
RHKsDSISLFFRKNWLRLTW





3270
RHLPsPPTLFFRKNWLRLTW





3271
RIGsDPLAYFFRKNWLRLTW





3272
RIIEtPPHRYFFRKNWLRLTW





3273
RIKLGDyHFYFFRKNWLRLTW





3274
RILFsPFFHFFRKNWLRLTW





3275
RILsATTSGIFLFFRKNWLRLTW





3276
RILsDVTHSAVFFRKNWLRLTW





3277
RILsGVVTKmFFRKNWLRLTW





3278
RILsGVVTKMFFRKNWLRLTW





3279
RILsGVVTKMKMFFRKNWLRLTW





3280
RIMsPMRTGNTYFFRKNWLRLTW





3281
RIQsPLNNKLFFRKNWLRLTW





3282
RIRsIEALLFFRKNWLRLTW





3283
RItSLIVHVFFRKNWLRLTW





3284
RITsPVHVSFFFRKNWLRLTW





3285
RIVsPKNSDLKFFRKNWLRLTW





3286
RIWsPTIGRFFRKNWLRLTW





3287
RIWSPtIGRFFRKNWLRLTW





3288
RIYsRIDRLEAFFRKNWLRLTW





3289
RKFsAPGQLFFRKNWLRLTW





3290
RKLsFTESLFFRKNWLRLTW





3291
RKLSFtESLFFRKNWLRLTW





3292
RKLsGDQITLFFRKNWLRLTW





3293
RKLsVALAFFFRKNWLRLTW





3294
RKLsVLLLLFFRKNWLRLTW





3295
RKNsFVmEYFFRKNWLRLTW





3296
RKNsFVMEYFFRKNWLRLTW





3297
RKNsLISSLFFRKNWLRLTW





3298
RKSsIIIRmFFRKNWLRLTW





3299
RLAsLFSSLFFRKNWLRLTW





3300
RLAsLMNLGMFFRKNWLRLTW





3301
RLAsYLEKVFFRKNWLRLTW





3302
RLDsELKELFFRKNWLRLTW





3303
RLDsGHVWKLFFRKNWLRLTW





3304
RLFsKELRcFFRKNWLRLTW





3305
RLFsKSIETLFFRKNWLRLTW





3306
RLFsSFLKRFFRKNWLRLTW





3307
RLIsLSEQNLFFRKNWLRLTW





3308
RLISLsEQNLFFRKNWLRLTW





3309
RLIsQIVSSFFRKNWLRLTW





3310
RLIsQIVSSITAFFRKNWLRLTW





3311
RLIsVVSHLFFRKNWLRLTW





3312
RLKsIEERQLLKFFRKNWLRLTW





3313
RLLQDsVDFSLFFRKNWLRLTW





3314
RLLQDsVDSLFFRKNWLRLTW





3315
RLLsAAENFFFRKNWLRLTW





3316
RLLsEKILGLFFRKNWLRLTW





3317
RLLsIKEAFRLFFRKNWLRLTW





3318
RLLsVNIRVFFRKNWLRLTW





3319
RLNsPPSSIYKFFRKNWLRLTW





3320
RLPLPsPALFFRKNWLRLTW





3321
RLPsDPFTHLFFRKNWLRLTW





3322
RLPsPTSPFSSLFFRKNWLRLTW





3323
RLPSsTLKRFFRKNWLRLTW





3324
RLPtVLLKLFFRKNWLRLTW





3325
RLQHSFsFFFRKNWLRLTW





3326
RLRsSVPGVFFRKNWLRLTW





3327
RLRSsVPGVFFRKNWLRLTW





3328
RLRsYEDmIFFRKNWLRLTW





3329
RLsPVPVPRFFRKNWLRLTW





3330
RLsSVSVTYFFRKNWLRLTW





3331
RLSsVSVTYFFRKNWLRLTW





3332
RLWtPPEDYRLFFRKNWLRLTW





3333
RLYKsEPELFFRKNWLRLTW





3334
RLYsVSYLLFFRKNWLRLTW





3335
RmIsHSELRKLFFRKNWLRLTW





3336
RMIsHSELRKLFFRKNWLRLTW





3337
RMIsKLEAQVFFRKNWLRLTW





3338
RmKsPFGSSFFFRKNWLRLTW





3339
RMKsPFGSSFFFRKNWLRLTW





3340
RmLsLRDQRLFFRKNWLRLTW





3341
RmYsFDDVLFFRKNWLRLTW





3342
RNAsLERVLFFRKNWLRLTW





3343
RPADSAQLLsLFFRKNWLRLTW





3344
RPARsVPSIAAFFRKNWLRLTW





3345
RPAsPALLLFFRKNWLRLTW





3346
RPAsPLMHIFFRKNWLRLTW





3347
RPASPsLQLFFRKNWLRLTW





3348
RPFHGISTVsLPNSLFFRKNWLRLTW





3349
RPFsKPEIALFFRKNWLRLTW





3350
RPFsREMDLFFRKNWLRLTW





3351
RPHLSGRKLsLFFRKNWLRLTW





3352
RPHtPTPGIFFRKNWLRLTW





3353
RPHtPTPGIYmFFRKNWLRLTW





3354
RPHTPtPGIYMFFRKNWLRLTW





3355
RPIsPRIGAFFRKNWLRLTW





3356
RPIsVIGGVSFFRKNWLRLTW





3357
RPItPVYTVFFRKNWLRLTW





3358
RPItPVYTVAFFRKNWLRLTW





3359
RPKLHHSLsFFFRKNWLRLTW





3360
RPKPSSsPVIFFRKNWLRLTW





3361
RPKPSsSPVIFFFRKNWLRLTW





3362
RPKPSSsPVIFFFRKNWLRLTW





3363
RPKPsSSPVIFAFFRKNWLRLTW





3364
RPKPSsSPVIFAFFRKNWLRLTW





3365
RPKPSSsPVIFAFFRKNWLRLTW





3366
RPKsTPELAFFFRKNWLRLTW





3367
RPKtPPPAPFFRKNWLRLTW





3368
RPLsKQLSAFFRKNWLRLTW





3369
RPLsLIQGPPFFRKNWLRLTW





3370
RPLsPFYLFFRKNWLRLTW





3371
RPLsPFYLSAFFRKNWLRLTW





3372
RPLsPGALQLFFRKNWLRLTW





3373
RPLsPILHIVFFRKNWLRLTW





3374
RPLsPKPSSPGFFRKNWLRLTW





3375
RPLsPKPSSPGSVLFFRKNWLRLTW





3376
RPLSPKPsSPGSVLFFRKNWLRLTW





3377
RPLsPTRLQPALFFRKNWLRLTW





3378
RPLtPRTPAFFRKNWLRLTW





3379
RPNsLVGITSAFFRKNWLRLTW





3380
RPNSPsPTALFFRKNWLRLTW





3381
RPNsSALETLFFRKNWLRLTW





3382
RPNSsALETLFFRKNWLRLTW





3383
RPPsPGLRGLLFFRKNWLRLTW





3384
RPQESRsLSPSHLFFRKNWLRLTW





3385
RPQESRSLsPSHLFFRKNWLRLTW





3386
RPQsPPAEAVIFFRKNWLRLTW





3387
RPQtPKEEAQALFFRKNWLRLTW





3388
RPRAFsHSGVHSLFFRKNWLRLTW





3389
RPRAFsIASSLFFRKNWLRLTW





3390
RPREVtVSLFFRKNWLRLTW





3391
RPRFMsSPVLFFRKNWLRLTW





3392
RPRFMSsPVLFFRKNWLRLTW





3393
RPRGPsPLVTmFFRKNWLRLTW





3394
RPRGPsPLVTMFFRKNWLRLTW





3395
RPRLQHsFSFFFRKNWLRLTW





3396
RPRLQHSFsFFFRKNWLRLTW





3397
RPRPSsVLRTLFFRKNWLRLTW





3398
RPRPVsPSSLLDTAIFFRKNWLRLTW





3399
RPRSIsVEEFFFRKNWLRLTW





3400
RPRSLSsPTVTLFFRKNWLRLTW





3401
RPRsPNmQDLFFRKNWLRLTW





3402
RPRsPPEPLRVFFRKNWLRLTW





3403
RPRSPtGPSNSFFFRKNWLRLTW





3404
RPRtLRTRLFFRKNWLRLTW





3405
RPsSAPDLmFFRKNWLRLTW





3406
RPsSAPDLMFFRKNWLRLTW





3407
RPSsAPDLmFFRKNWLRLTW





3408
RPSsAPDLMFFRKNWLRLTW





3409
RPsSGFYELFFRKNWLRLTW





3410
RPsSGQDLFFFRKNWLRLTW





3411
RPSsGQDLFFFRKNWLRLTW





3412
RPSsLRQYLFFRKNWLRLTW





3413
RPSsPLIDIKPFFRKNWLRLTW





3414
RPsSPVHVAFFFRKNWLRLTW





3415
RPSsPVHVAFFFRKNWLRLTW





3416
RPSsPVTVTALFFRKNWLRLTW





3417
RPSsRVALmVLFFRKNWLRLTW





3418
RPSsRVALMVLFFRKNWLRLTW





3419
RPStPHTITLFFRKNWLRLTW





3420
RPsTPTINVLFFRKNWLRLTW





3421
RPStPTINVLFFRKNWLRLTW





3422
RPSTPtINVLFFRKNWLRLTW





3423
RPtSFADELFFRKNWLRLTW





3424
RPTsISWDGLFFRKNWLRLTW





3425
RPTSIsWDGLFFRKNWLRLTW





3426
RPTsPRLLTLFFRKNWLRLTW





3427
RPVDPRRRsLFFRKNWLRLTW





3428
RPVsEMFSLFFRKNWLRLTW





3429
RPVsMDARIQVFFRKNWLRLTW





3430
RPVsPGKDITAFFRKNWLRLTW





3431
RPVStDFAQYFFRKNWLRLTW





3432
RPVtPITNFFFRKNWLRLTW





3433
RPVtPPRTAFFRKNWLRLTW





3434
RPwsNSRGLFFRKNWLRLTW





3435
RPwsPAVSAFFRKNWLRLTW





3436
RPYPsPGAVLFFRKNWLRLTW





3437
RQAsIELPSMAFFRKNWLRLTW





3438
RQAsIELPSmAVFFRKNWLRLTW





3439
RQAsIELPSmAVAFFRKNWLRLTW





3440
RQAsIELPSmAVASTFFRKNWLRLTW





3441
RQAsIELPSMAVASTFFRKNWLRLTW





3442
RQASLsISVFFRKNWLRLTW





3443
RQFDEESLEsFFFRKNWLRLTW





3444
RQFTSSSsIFFRKNWLRLTW





3445
RQHFsPLSLFFRKNWLRLTW





3446
RQIQPsPPwSYFFRKNWLRLTW





3447
RQIQPsPPWSYFFRKNWLRLTW





3448
RQIsIRGIVGVFFRKNWLRLTW





3449
RQISISEPQAFFRKNWLRLTW





3450
RQISISEPQAFFFRKNWLRLTW





3451
RQISISEPQAFLFFRKNWLRLTW





3452
RQIsISEPQAFLFFFRKNWLRLTW





3453
RQIsPEEFEYFFRKNWLRLTW





3454
RQKsPLFQFAFFRKNWLRLTW





3455
RQPsEEEIIFFRKNWLRLTW





3456
RQPsEEEIIKLFFRKNWLRLTW





3457
RQPsWDPSPVFFRKNWLRLTW





3458
RQRSLsTSGESLYFFRKNWLRLTW





3459
RQVsEDPDIDSLFFRKNWLRLTW





3460
RRAsLSDIGFFFRKNWLRLTW





3461
RRFRFPsGAELFFRKNWLRLTW





3462
RRFsDFLGLFFRKNWLRLTW





3463
RRFSFsGNTLFFRKNWLRLTW





3464
RRFsGLLNFFRKNWLRLTW





3465
RRFsGLLNcFFRKNWLRLTW





3466
RRFsGLLNCFFRKNWLRLTW





3467
RRFsGLSAELFFRKNWLRLTW





3468
RRFsLDTDYFFRKNWLRLTW





3469
RRFsPPRRMLFFRKNWLRLTW





3470
RRFsVTLRLFFRKNWLRLTW





3471
RRFtEIYEFFFRKNWLRLTW





3472
RRFtPPSTALFFRKNWLRLTW





3473
RRGsFDAFFRKNWLRLTW





3474
RRGsFDATFFRKNWLRLTW





3475
RRGsFDATGFFRKNWLRLTW





3476
RRGsFDATGSGFFRKNWLRLTW





3477
RRGsFDATGSGFFFRKNWLRLTW





3478
RRGsFDATGSGFSMFFRKNWLRLTW





3479
RRGsFDATGSGFSmTFFFRKNWLRLT



W





3480
RRGsFDATGSGFSMTFFFRKNWLRLT



W





3481
RRGsFEVTLLFFRKNWLRLTW





3482
RRGsGPEIFTFFFRKNWLRLTW





3483
RRGsPEMPFYFFRKNWLRLTW





3484
RRIDIsPSTFRKFFRKNWLRLTW





3485
RRIDISPsTLRKFFRKNWLRLTW





3486
RRISLtKRLFFRKNWLRLTW





3487
RRLDRRwtLFFRKNWLRLTW





3488
RRLDRRWtLFFRKNWLRLTW





3489
RRLsFQAEYWFFRKNWLRLTW





3490
RRLsLFLVLFFRKNWLRLTW





3491
RRLsVLVDDYFFRKNWLRLTW





3492
RRMsVGDRAGFFRKNWLRLTW





3493
RRMsVGDRAGSLPNYFFRKNWLRLTW





3494
RRNsLRIIFFFRKNWLRLTW





3495
RRPsQNAISFFFFRKNWLRLTW





3496
RRPtLTTFFFFRKNWLRLTW





3497
RRsDSLLSFFFRKNWLRLTW





3498
RRSDsLLSFFFRKNWLRLTW





3499
RRSIIsPNFFFRKNWLRLTW





3500
RRsSFSMEEGDVLFFRKNWLRLTW





3501
RRSsFSMEEGDVLFFRKNWLRLTW





3502
RRsSIPITVFFRKNWLRLTW





3503
RRSsISSWLFFRKNWLRLTW





3504
RRsSLLSLmFFRKNWLRLTW





3505
RRsSLLSLMFFRKNWLRLTW





3506
RRSsLLSLmFFRKNWLRLTW





3507
RRsSYLLAIFFRKNWLRLTW





3508
RRSsYLLAIFFRKNWLRLTW





3509
RRsTGVSFWFFRKNWLRLTW





3510
RRStGVSFWFFRKNWLRLTW





3511
RRTsIHDFLFFRKNWLRLTW





3512
RRVsLSEIGFFFRKNWLRLTW





3513
RRVsSNGIFDLFFRKNWLRLTW





3514
RRVSsNGIFDLFFRKNWLRLTW





3515
RRYsDFAKLFFRKNWLRLTW





3516
RSELLsFIKFFRKNWLRLTW





3517
RSFsADNFIGIQRFFRKNWLRLTW





3518
RSFsGLIKRFFRKNWLRLTW





3519
RSFsMHDLTTIFFRKNWLRLTW





3520
RSFsPKSPLELFFRKNWLRLTW





3521
RSFsPTmKVFFRKNWLRLTW





3522
RSFSPtMKVFFRKNWLRLTW





3523
RSFtPLSIFFRKNWLRLTW





3524
RSFtPLSILKFFRKNWLRLTW





3525
RSHsPPLKLFFRKNWLRLTW





3526
RSIRDsGYIDFFRKNWLRLTW





3527
RSIRDsGYIDcwFFRKNWLRLTW





3528
RSIRDsGYIDcWFFRKNWLRLTW





3529
RSISAsDLTFFFRKNWLRLTW





3530
RSIsNEGLTLFFRKNWLRLTW





3531
RSIsPLLFFFRKNWLRLTW





3532
RSIsPWLARFFRKNWLRLTW





3533
RSIsQSSTDSYFFRKNWLRLTW





3534
RSIsSLLRFFFRKNWLRLTW





3535
RSIsTPTcLFFRKNWLRLTW





3536
RSKsVIEQVFFRKNWLRLTW





3537
RSKsVIEQVSWFFRKNWLRLTW





3538
RSLsFSDEMFFRKNWLRLTW





3539
RSLsPFRRHFFRKNWLRLTW





3540
RSLsPIIGKDVLFFRKNWLRLTW





3541
RSLsPILPGRFFRKNWLRLTW





3542
RSLsPmSGLFFRKNWLRLTW





3543
RSLsPMSGLFFRKNWLRLTW





3544
RSLsPSSNSAFFFRKNWLRLTW





3545
RsLSQELVGVFFRKNWLRLTW





3546
RsLSVEIVYFFRKNWLRLTW





3547
RSLsVGSEFFFRKNWLRLTW





3548
RSLsVPVDLFFRKNWLRLTW





3549
RSLsVPVDLSRWFFRKNWLRLTW





3550
RSLtHPPTIFFRKNWLRLTW





3551
RSmDSVLtLFFRKNWLRLTW





3552
RSMDSVLtLFFRKNWLRLTW





3553
RSNsPLPSIFFRKNWLRLTW





3554
RSPsFGEDYYFFRKNWLRLTW





3555
RSPsQDFSFFFRKNWLRLTW





3556
RSQsLPNSLFFRKNWLRLTW





3557
RSRsAPPNLWFFRKNWLRLTW





3558
RSRsFDYNYFFRKNWLRLTW





3559
RSRsFDYNYRFFRKNWLRLTW





3560
RSRsFSGLIKRFFRKNWLRLTW





3561
RSRSFsGLIKRFFRKNWLRLTW





3562
RSRsPFSTTRFFRKNWLRLTW





3563
RSRsPLELEPEAKFFRKNWLRLTW





3564
RSRsPLGFYVFFRKNWLRLTW





3565
RSRsPLLKFFFRKNWLRLTW





3566
RSRsPSDSAAYFFFRKNWLRLTW





3567
RSRsVPVSFFFRKNWLRLTW





3568
RSSsFKDFAKFFRKNWLRLTW





3569
RSSsFSDTLFFRKNWLRLTW





3570
RSsSFVLPKFFRKNWLRLTW





3571
RSSsFVLPKFFRKNWLRLTW





3572
RsSSFVLPKLFFRKNWLRLTW





3573
RSsSFVLPKLFFRKNWLRLTW





3574
RSSsFVLPKLFFRKNWLRLTW





3575
RsSSLSDFSwFFRKNWLRLTW





3576
RsSSLSDFSWFFRKNWLRLTW





3577
RSsSLSDFSwFFRKNWLRLTW





3578
RSsSLSDFSWFFRKNWLRLTW





3579
RSSsLSDFSwFFRKNWLRLTW





3580
RSSsLSDFSWFFRKNWLRLTW





3581
RsSSPFLSKFFRKNWLRLTW





3582
RSsSPFLSKFFRKNWLRLTW





3583
RSSsPPILTKFFRKNWLRLTW





3584
RSsSTELLSHYFFRKNWLRLTW





3585
RSSsTELLSHYFFRKNWLRLTW





3586
RSSsWGRTYFFRKNWLRLTW





3587
RSStPLPTIFFRKNWLRLTW





3588
RsTSLSLKYFFRKNWLRLTW





3589
RStSLSLKYFFRKNWLRLTW





3590
RSTsLSLKYFFRKNWLRLTW





3591
RSVsFKLLERWFFRKNWLRLTW





3592
RSVsPVQDLFFRKNWLRLTW





3593
RSVsVATGLFFRKNWLRLTW





3594
RSWsPPPEVSRFFRKNWLRLTW





3595
RSYRTDIsMFFRKNWLRLTW





3596
RTAsPPALPKFFRKNWLRLTW





3597
RTFsDESNVLFFRKNWLRLTW





3598
RtFSLDTILFFRKNWLRLTW





3599
RTFsLDTILSSYFFRKNWLRLTW





3600
RTFSPtYGLFFRKNWLRLTW





3601
RtHSLLLLLFFRKNWLRLTW





3602
RtISAQDTLAYFFRKNWLRLTW





3603
RTIsAQDTLAYFFRKNWLRLTW





3604
RTIsNPEVVmKFFRKNWLRLTW





3605
RTIsNPEVVMKFFRKNWLRLTW





3606
RTKsFLNYYFFRKNWLRLTW





3607
RTLsESFSRIALKFFRKNWLRLTW





3608
RTLsGSILDVYFFRKNWLRLTW





3609
RtmSEAALVRKFFRKNWLRLTW





3610
RtMSEAALVRKFFRKNWLRLTW





3611
RTmsPIQVLFFRKNWLRLTW





3612
RTMsPIQVLFFRKNWLRLTW





3613
RTPsPARPALFFRKNWLRLTW





3614
RTRLsPPRAFFRKNWLRLTW





3615
RTVsPAHVLFFRKNWLRLTW





3616
RTYsFTSAmFFRKNWLRLTW





3617
RTYsFTSAMFFRKNWLRLTW





3618
RVASPtSGVFFRKNWLRLTW





3619
RVDSLVsLFFRKNWLRLTW





3620
RVDsTTcLFFFRKNWLRLTW





3621
RVDStTcLFFFRKNWLRLTW





3622
RVDSTtcLFFFRKNWLRLTW





3623
RVIsLEDFMEKFFRKNWLRLTW





3624
RVKTPtSQSYFFRKNWLRLTW





3625
RVKVDGPRsPSYFFRKNWLRLTW





3626
RVKVDGPRSPsYFFRKNWLRLTW





3627
RVLsPLmSRFFRKNWLRLTW





3628
RVLsPLMSRFFRKNWLRLTW





3629
RVPsINQKIFFRKNWLRLTW





3630
RVRsFLRGLPFFRKNWLRLTW





3631
RVRsPGTGAFFFRKNWLRLTW





3632
RVsSLTLHLFFRKNWLRLTW





3633
RVSsLTLHLFFRKNWLRLTW





3634
RVSSLtLHLFFRKNWLRLTW





3635
RVVLtPLKVFFRKNWLRLTW





3636
RVVsPGIDLFFRKNWLRLTW





3637
RVYsLDDIRRYFFRKNWLRLTW





3638
RVYsRFEVFFFRKNWLRLTW





3639
RVYYsPPVARRFFRKNWLRLTW





3640
RWNsKENLLFFRKNWLRLTW





3641
RYARYsPRQRFFRKNWLRLTW





3642
RYDsRTTIFFFRKNWLRLTW





3643
RYFKtPRKFFFRKNWLRLTW





3644
RYHsLAPmYYFFRKNWLRLTW





3645
RYHsLAPMYYFFRKNWLRLTW





3646
RYtNRVVTLFFRKNWLRLTW





3647
SAFsSRGSLSLFFRKNWLRLTW





3648
sAISPTPEIFFRKNWLRLTW





3649
SAIsPTPEIFFRKNWLRLTW





3650
SAYGGLTsPGLSYFFRKNWLRLTW





3651
SEAsLASALFFRKNWLRLTW





3652
SEFKAmDsIFFRKNWLRLTW





3653
SEFsDVDKLFFRKNWLRLTW





3654
SEIsPIKGSVRFFRKNWLRLTW





3655
SELRsPRISYFFRKNWLRLTW





3656
SELtPSESLFFRKNWLRLTW





3657
SELTPsESLFFRKNWLRLTW





3658
SEsSIKKKFLFFRKNWLRLTW





3659
SESsIKKKFLFFRKNWLRLTW





3660
SFDsREASFFFRKNWLRLTW





3661
SFLsQDESHDHSFFFRKNWLRLTW





3662
sGEGDFLAEGGGVRFFRKNWLRLTW





3663
SGFRsPHLwFFRKNWLRLTW





3664
SGFRsPHLWFFRKNWLRLTW





3665
SIDIsQDKLFFRKNWLRLTW





3666
sIDSPKSYIFFRKNWLRLTW





3667
SIFRtPISKFFRKNWLRLTW





3668
SIIKEKtVFFRKNWLRLTW





3669
SIIsPKVKMALFFRKNWLRLTW





3670
SIIsPNFSFFFRKNWLRLTW





3671
SILsRTPSVFFRKNWLRLTW





3672
sIPSLVDGFFFRKNWLRLTW





3673
SIPsLVDGFFFRKNWLRLTW





3674
SIPTVsGQIFFRKNWLRLTW





3675
SISsIDRELFFRKNWLRLTW





3676
SISsmEVNVFFRKNWLRLTW





3677
SIsTLVTLFFRKNWLRLTW





3678
SIStLVTLFFRKNWLRLTW





3679
SItSLEAIIFFRKNWLRLTW





3680
SIVsPRKLPALFFRKNWLRLTW





3681
SKMAFLtRVAFFRKNWLRLTW





3682
SLAsKVTRLFFRKNWLRLTW





3683
SLAsLLAKVFFRKNWLRLTW





3684
SLDsPGPEKmALFFRKNWLRLTW





3685
SLDsPGPEKMALFFRKNWLRLTW





3686
SLFGsPVAKFFRKNWLRLTW





3687
SLFHtPKFVFFRKNWLRLTW





3688
SLFSsEESNLGAFFRKNWLRLTW





3689
SLLsELQHAFFRKNWLRLTW





3690
SLLsLSATVFFRKNWLRLTW





3691
SLLsVSHALFFRKNWLRLTW





3692
SLLtPVRLPSIFFRKNWLRLTW





3693
SLmsGTLESLFFRKNWLRLTW





3694
SLmSGtLESLFFRKNWLRLTW





3695
SLMSGtLESLFFRKNWLRLTW





3696
SLSsERYYLFFRKNWLRLTW





3697
SLsSLRAHLEYFFRKNWLRLTW





3698
SLSsLRAHLEYFFRKNWLRLTW





3699
SmKsPLYLVSRFFRKNWLRLTW





3700
SMKsPLYLVSRFFRKNWLRLTW





3701
SPAARSLsLFFRKNWLRLTW





3702
SPAsPLKELFFRKNWLRLTW





3703
SPDIsPPIFRRFFRKNWLRLTW





3704
SPFKRQLsFFRKNWLRLTW





3705
SPFLSKRsLFFRKNWLRLTW





3706
SPFSSRsPSLFFRKNWLRLTW





3707
SPGsPWKTKLFFRKNWLRLTW





3708
sPHSPFYQLFFRKNWLRLTW





3709
SPHsPFYQLFFRKNWLRLTW





3710
SPIsDEEERLFFRKNWLRLTW





3711
SPIsPRTQDALFFRKNWLRLTW





3712
SPIsPTRQDALFFRKNWLRLTW





3713
SPITSsPPKWFFRKNWLRLTW





3714
SPKPPtRSPFFRKNWLRLTW





3715
SPKPPTRsPFFRKNWLRLTW





3716
SPPsPARWSLFFRKNWLRLTW





3717
SPRAGsPFFFRKNWLRLTW





3718
SPRAGsPFSPPPSSSSLFFRKNWLRL



TW





3719
SPRLVsRSSSVLFFRKNWLRLTW





3720
SPRPPNSPsIFFRKNWLRLTW





3721
SPRPPNsPSISIFFRKNWLRLTW





3722
SPRPtSAPAIFFRKNWLRLTW





3723
SPRPTsAPAIFFRKNWLRLTW





3724
SPRRPsRVSEFFFRKNWLRLTW





3725
SPRRPsRVSEFLFFRKNWLRLTW





3726
sPRSPISPELFFRKNWLRLTW





3727
SPRsPISPELFFRKNWLRLTW





3728
sPRSPSTTYLFFRKNWLRLTW





3729
SPRsPTTTLFFRKNWLRLTW





3730
SPRsPVNKTTLFFRKNWLRLTW





3731
sPRSPVPTTLFFRKNWLRLTW





3732
SPRsPVPTTLFFRKNWLRLTW





3733
sPRTPPPLTVFFRKNWLRLTW





3734
SPRtPPPLTVFFRKNWLRLTW





3735
SPRTPtPFKHALFFRKNWLRLTW





3736
SPRtPVSPVKFFFRKNWLRLTW





3737
SPsPLPVALFFRKNWLRLTW





3738
SPsPmDPHMFFRKNWLRLTW





3739
SPsPMDPHmFFRKNWLRLTW





3740
SPsPMDPHMFFRKNWLRLTW





3741
SPtSPDYSLFFRKNWLRLTW





3742
SPtSPFSSLFFRKNWLRLTW





3743
SPTsPFSSLFFRKNWLRLTW





3744
SPVNKVRRVsFFFRKNWLRLTW





3745
SPVsPKSLAFFFRKNWLRLTW





3746
SPVsPmKELFFRKNWLRLTW





3747
SQDsPIFmFFRKNWLRLTW





3748
SQDsPIFMFFRKNWLRLTW





3749
SQILRTPsLFFRKNWLRLTW





3750
SRFHsPSTTWFFRKNWLRLTW





3751
SRFsGGFGAFFRKNWLRLTW





3752
SRFsGGFGARDYFFRKNWLRLTW





3753
SRHsGPFFTFFFRKNWLRLTW





3754
SRKEsYSVYVYFFRKNWLRLTW





3755
SRKsFVFELFFRKNWLRLTW





3756
SRLsLRRFFRKNWLRLTW





3757
SRLsLRRSLFFRKNWLRLTW





3758
SRPSmsPTPLFFRKNWLRLTW





3759
SRPSMsPTPLFFRKNWLRLTW





3760
SRRsIFEMYFFRKNWLRLTW





3761
SRSsPLKLFFRKNWLRLTW





3762
SSIsPSTLTLKFFRKNWLRLTW





3763
SSLsGEELVTKFFRKNWLRLTW





3764
SSLSsPLNPKFFRKNWLRLTW





3765
SSSsPFKFKFFRKNWLRLTW





3766
STAsAITPSVSRFFRKNWLRLTW





3767
STGGGTVIsRFFRKNWLRLTW





3768
STsLEKNNVFFRKNWLRLTW





3769
SVFsPSFGLKFFRKNWLRLTW





3770
SVIsDDSVLFFRKNWLRLTW





3771
SVIsGISSRFFRKNWLRLTW





3772
SVISsPLLKFFRKNWLRLTW





3773
SVLsPLLNKFFRKNWLRLTW





3774
SVLsPTSWEKFFRKNWLRLTW





3775
SVLsYTSVRFFRKNWLRLTW





3776
SVLtPLLLRFFRKNWLRLTW





3777
SVPEFPLsPPKKFFRKNWLRLTW





3778
SVQsDQGYISRFFRKNWLRLTW





3779
SVSsLEVHFFFRKNWLRLTW





3780
SVTsPIKmKFFRKNWLRLTW





3781
SVTsPIKMKFFRKNWLRLTW





3782
SVVsFDKVKEPRFFRKNWLRLTW





3783
SVVsGSEMSGKYFFRKNWLRLTW





3784
SVYsPSGPVNRFFRKNWLRLTW





3785
SVYSPsGPVNRFFRKNWLRLTW





3786
SYPsPVPTSFFFRKNWLRLTW





3787
SYVTTSTRTYsLGFFRKNWLRLTW





3788
SYYsPSIGFSYFFRKNWLRLTW





3789
TAIsPPLSVFFRKNWLRLTW





3790
TELPKRLsLFFRKNWLRLTW





3791
TESsPGSRQIQLwFFRKNWLRLTW





3792
TESsPGSRQIQLWFFRKNWLRLTW





3793
TEVsPSRTIFFRKNWLRLTW





3794
THALPEsPRLFFRKNWLRLTW





3795
THDsPFcLFFRKNWLRLTW





3796
THIsPNAIFFFRKNWLRLTW





3797
THIsPNAIFKAFFRKNWLRLTW





3798
TIFsPEGRLYFFRKNWLRLTW





3799
TImsPAVLKFFRKNWLRLTW





3800
TIMsPAVLKFFRKNWLRLTW





3801
TIRSPtTVLFFRKNWLRLTW





3802
TLAsPSVFKFFRKNWLRLTW





3803
TLLAsPmLKFFRKNWLRLTW





3804
TLLsAAHEVELFFRKNWLRLTW





3805
TLLsPKHKYFFRKNWLRLTW





3806
TLPsPDKLPGFFFRKNWLRLTW





3807
TLSCPVtEVIFFRKNWLRLTW





3808
TLsSIRHMIFFRKNWLRLTW





3809
TLSsIRHmIFFRKNWLRLTW





3810
TLSsIRHMIFFRKNWLRLTW





3811
TLYPRSFsVFFRKNWLRLTW





3812
TmFLRETsLFFRKNWLRLTW





3813
TMFLREtSLFFRKNWLRLTW





3814
TMFLRETsLFFRKNWLRLTW





3815
TmLsPREKIFYYFFRKNWLRLTW





3816
TMLsPREKIFYYFFRKNWLRLTW





3817
TPAGSARGsPTRPNPPFFRKNWLRLT



W





3818
TPHtPKSLLFFRKNWLRLTW





3819
TPIsPGRASGmTTLFFRKNWLRLTW





3820
TPIsPGRASGMTTLFFRKNWLRLTW





3821
tPPSSEKLVSVMFFRKNWLRLTW





3822
TPQPsKDTLLFFRKNWLRLTW





3823
TPsPARPALFFRKNWLRLTW





3824
TPVsPVKFFFRKNWLRLTW





3825
TQRKFsLQFFFRKNWLRLTW





3826
TRDsLLIHLFFRKNWLRLTW





3827
TSEtPQPPRFFRKNWLRLTW





3828
TSIsPALARFFRKNWLRLTW





3829
TSVGsPSNTIGRFFRKNWLRLTW





3830
TSYNSISSVVsRFFRKNWLRLTW





3831
TTEVIRKGsITEYFFRKNWLRLTW





3832
tTGSPTEFLFFRKNWLRLTW





3833
TtGSPTEFLFFRKNWLRLTW





3834
TTGsPTEFLFFRKNWLRLTW





3835
TVFsPDGHLFFFRKNWLRLTW





3836
TVFSPtLPAAFFRKNWLRLTW





3837
TVFsPTLPAARFFRKNWLRLTW





3838
TVFtPVEEKFFRKNWLRLTW





3839
TVKQKYLsFFFRKNWLRLTW





3840
TVNsPATYKFFRKNWLRLTW





3841
TVNsPATYKFFFRKNWLRLTW





3842
TVStPPPFQGRFFRKNWLRLTW





3843
TVsTVGISIFFRKNWLRLTW





3844
TVVsPRALELFFRKNWLRLTW





3845
TVYSsEEAELLKFFRKNWLRLTW





3846
TYDDRAYSsFFFRKNWLRLTW





3847
TYVsSFYHAFFFRKNWLRLTW





3848
VAKRNsLKELWFFRKNWLRLTW





3849
VARsPLKEFFFRKNWLRLTW





3850
VEHsPFSSFFFRKNWLRLTW





3851
VELsPARSwFFRKNWLRLTW





3852
VELsPARSWFFRKNWLRLTW





3853
VELsPLKGSVSWFFRKNWLRLTW





3854
VETsFRKLSFFFRKNWLRLTW





3855
VETSFRKLsFFFRKNWLRLTW





3856
VIDsQELSKFFRKNWLRLTW





3857
VIKsPSWQRFFRKNWLRLTW





3858
VImsIRTKLFFRKNWLRLTW





3859
VIMsIRTKLFFRKNWLRLTW





3860
VLAsPLKTGRFFRKNWLRLTW





3861
VLFSsPPQmFFRKNWLRLTW





3862
VLGsQEALHPVFFRKNWLRLTW





3863
VLPSQVYsLFFRKNWLRLTW





3864
VmDsPVHLFFRKNWLRLTW





3865
VmFRtPLASVFFRKNWLRLTW





3866
VPFKRLsVVFFFRKNWLRLTW





3867
VPKGPIHsPVELFFRKNWLRLTW





3868
VPKKPPPsPFFRKNWLRLTW





3869
VPNEEDPsLFFRKNWLRLTW





3870
VPRsPFKVKVLFFRKNWLRLTW





3871
VPRsPVIKIFFRKNWLRLTW





3872
VPRtPVGKFFFRKNWLRLTW





3873
VPSsPLRKAFFRKNWLRLTW





3874
VPTsPKGRLLFFRKNWLRLTW





3875
VRKsRAWVLFFRKNWLRLTW





3876
VRTPSVQsLFFRKNWLRLTW





3877
VSFsPTDHSLFFRKNWLRLTW





3878
VSSsPRELLFFRKNWLRLTW





3879
VVSsPKLAPKFFRKNWLRLTW





3880
VYIPmsPGAHHFFFRKNWLRLTW





3881
VYIPMsPGAHHFFFRKNWLRLTW





3882
VYLPTHtSLFFRKNWLRLTW





3883
VYLPTHTsLFFRKNWLRLTW





3884
VYLPTHtSLLFFRKNWLRLTW





3885
VYLPTHTsLLFFRKNWLRLTW





3886
VYTsVQAQYFFRKNWLRLTW





3887
WEDRPStPTILFFRKNWLRLTW





3888
WEFGKRDsLFFRKNWLRLTW





3889
WPRsPGRAFLFFRKNWLRLTW





3890
WVIGsPEILRFFRKNWLRLTW





3891
YAFsPKIGRFFRKNWLRLTW





3892
yEKIHLDFLFFRKNWLRLTW





3893
YEVEPYsPGLFFRKNWLRLTW





3894
YHLsPRAFLFFRKNWLRLTW





3895
YILDSsPEKLFFRKNWLRLTW





3896
YLRsVGDGETVFFRKNWLRLTW





3897
YLVsPITGEKIFFRKNWLRLTW





3898
YPDPHsPFAFFRKNWLRLTW





3899
YPFLDsPNKYSLFFRKNWLRLTW





3900
YPSFRRSsLFFRKNWLRLTW





3901
YPtPYPDELFFRKNWLRLTW





3902
YQLsPTKLPSINFFRKNWLRLTW





3903
YQRPFSPsAYFFRKNWLRLTW





3904
YQYsDQGIDYFFRKNWLRLTW





3905
YRLsPEPTPLFFRKNWLRLTW





3906
YRPsYSYDYFFRKNWLRLTW





3907
YRPsYSYDYEFDFFRKNWLRLTW





3908
YRYDGQHFsLFFRKNWLRLTW





3909
YRYsLEKALFFRKNWLRLTW





3910
YSLDsPGPEKmALFFRKNWLRLTW





3911
YSLDsPGPEKMALFFRKNWLRLTW





3912
YSLsPSKSYKYFFRKNWLRLTW





3913
YSmsPGAMRFFRKNWLRLTW





3914
YSMsPGAmRFFRKNWLRLTW





3915
YSMsPGAMRFFRKNWLRLTW





3916
YVKLTPVsLFFRKNWLRLTW





3917
YVSsPDPQLFFRKNWLRLTW





3918
YYFsPSGKKFFFRKNWLRLTW





3919
yYISPRITFFFRKNWLRLTW





4073
DIAsLVGHEFFFRKNWLRLTW





4074
DIVsEYTHYFFRKNWLRLTW





4075
DSADLPPPsALFFRKNWLRLTW





4076
DVIDsQELSKVSREFFFRKNWLRLTW





4077
ETRSPsPISIFFRKNWLRLTW





4078
FKmIRSQsLFFRKNWLRLTW





4079
GAVsPGALRFFRKNWLRLTW





4080
GLPsPRGPGLFFRKNWLRLTW





4081
GRILsGVVTKFFRKNWLRLTW





4082
GRMIRAEsGPDLRYFFRKNWLRLTW





4083
GRmIRAEsGPDLRYFFRKNWLRLTW





4084
HPDGtPPKLFFRKNWLRLTW





4085
HPHLRKVsVFFRKNWLRLTW





4086
HRRIDIsPSTLFFRKNWLRLTW





4087
KAsSLISLLFFRKNWLRLTW





4088
KASsLISLLFFRKNWLRLTW





4089
KIPsAVSTVSMFFRKNWLRLTW





4090
KRFsMVVQDGIVKFFRKNWLRLTW





4091
KRFsmVVQDGIVKFFRKNWLRLTW





4092
KRFStEEFVLLFFRKNWLRLTW





4093
KRIsISISFFRKNWLRLTW





4094
KRIsISTSGFFRKNWLRLTW





4095
KRIsISTSGGFFRKNWLRLTW





4096
KRLsLDSSLVEYFFRKNWLRLTW





4097
KRLsLPADIRLFFRKNWLRLTW





4098
KRTsKYFSLFFRKNWLRLTW





4099
LPRsSSMAAGLFFRKNWLRLTW





4100
LPRSsSMAAGLFFRKNWLRLTW





4101
LQHsFSFAGFFFRKNWLRLTW





4102
LtSKLSTKDFFRKNWLRLTW





4103
NPTMLRTHsLFFRKNWLRLTW





4104
NRsSPVHIIFFRKNWLRLTW





4105
QVLPKtVKLFFFRKNWLRLTW





4106
RLPSPtSPFSSLFFRKNWLRLTW





4107
RPKLHHsLSFFFRKNWLRLTW





4108
RPRsDSLILFFRKNWLRLTW





4109
RQPswDPSPVFFRKNWLRLTW





4110
RRAsAPLPGLFFRKNWLRLTW





4111
RRASLsEIGFFRKNWLRLTW





4112
RRAsLSEIGFFRKNWLRLTW





4113
RRFsADEQFFFFRKNWLRLTW





4114
RRFsFSANFYFFRKNWLRLTW





4115
RRFsPPSSSLFFRKNWLRLTW





4116
RRIDIsPSFFRKNWLRLTW





4117
RRIsIVENcFFFRKNWLRLTW





4118
RRLPIFsRLSIFFRKNWLRLTW





4119
RRLsAIFLRLFFRKNWLRLTW





4120
RRLsFLVSYIFFRKNWLRLTW





4121
RRLsFTLERLFFRKNWLRLTW





4122
RRLsIEGNIAVFFRKNWLRLTW





4123
RRLsPPTLLFFRKNWLRLTW





4124
RSFSPtmKVFFRKNWLRLTW





4125
RSsSFTFHIFFRKNWLRLTW





4126
RSSsFTFHIFFRKNWLRLTW





4127
RtAATEVSLFFRKNWLRLTW





4128
RVDsTTCLFFFRKNWLRLTW





4129
RVDsTTcLFPFFRKNWLRLTW





4130
RVPsEHPYLFFRKNWLRLTW





4131
SAITPSVSRTsFFFRKNWLRLTW





4132
SEGsEPALLHFFRKNWLRLTW





4133
SIAsPDVKLNLFFRKNWLRLTW





4134
SIKsDVPVYFFRKNWLRLTW





4135
SLALtPPQAFFRKNWLRLTW





4136
SLKsRLRFFRKNWLRLTW





4137
SLPsPHPVRYFFRKNWLRLTW





4138
SPRPSPVPKPsPPLFFRKNWLRLTW





4139
SRFsSGGAFFRKNWLRLTW





4140
SRIVRTPsLFFRKNWLRLTW





4141
SRTSFTSVsRFFRKNWLRLTW





4142
TMPTsLPNLFFRKNWLRLTW





4143
TRLsPIAPAPGFFFRKNWLRLTW





4144
TSNsQKYmSFFFRKNWLRLTW





4145
TSTSRYLsLFFRKNWLRLTW





4146
VKTsGSSDRLFFRKNWLRLTW





4147
NIKsPALAFFRKNWLRLTW





4148
LsPRAVSTTFFFRKNWLRLTW





4195
AHDPSGMFRSQsFFFRKNWLRLTW





4196
RVAsPAYSLFFRKNWLRLTW





4197
RRWtLGGMVNRFFRKNWLRLTW





4198
SIPSTLVsFFFRKNWLRLTW





4199
RRGsYPFIDFFFRKNWLRLTW





4200
LtLDQAYSYFFRKNWLRLTW





4201
SPPsPVEREmFFRKNWLRLTW





4202
SPPsPVEREMFFRKNWLRLTW





4203
LYVLsALLIFFRKNWLRLTW





4204
RPRsLSSPTVFFRKNWLRLTW





4205
LPIFNRIsVFFRKNWLRLTW





4206
IPRYHSQsPSmFFRKNWLRLTW





4207
SPLVRRPsLFFRKNWLRLTW





4208
EAPKVSRsLFFRKNWLRLTW





4209
SLDSPsYVLYFFRKNWLRLTW





4210
REYsPPYAPFFRKNWLRLTW





4211
YGYEGSEsIFFRKNWLRLTW





4212
RPSsLPLDFFFRKNWLRLTW





4213
RPsSLPLDFFFRKNWLRLTW





4214
TPItPLKDGFFFRKNWLRLTW





4215
KRFsFKKSFKLFFRKNWLRLTW





4216
KRNsRLGFLYFFRKNWLRLTW





4217
RRAsAILPGVLFFRKNWLRLTW





‘s’, ‘t, and ‘y’ stand for phosphoserine, phosphothreonine, and phosphotyrosine, respectively.


‘m’ stands for oxidized methionine.


‘w’ stands for oxidized tryptophan.


‘c’ stands for cysteinylated cysteine.






In certain embodiments, the instant disclosure provides: an antigenic polypeptide comprising an MHC-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171; and an HSP-binding peptide comprising the amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein X1 is omitted, N, F, or Q; X2 is W, L, or F; X3 is L or I; X4 is R, L, or K; X5 is L, W, or I; X6 is T, L, F, K, R, or W; and X7 is W, G, K, or F.


In certain embodiments, the HSP-binding peptide comprises the amino acid sequence of:

    • (a) X1LX2LTX3 (SEQ ID NO: 2), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;
    • (b) NX1LX2LTX3 (SEQ ID NO: 3), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;
    • (c) WLX1LTX2 (SEQ ID NO: 4), wherein X1 is R or K; and X2 is W or G;
    • (d) NWLX1LTX2 (SEQ ID NO: 5), wherein X1 is R or K; and X2 is W or G; or
    • (e) NWX1X2X3X4X5 (SEQ ID NO: 6), wherein X1 is L or I; X2 is L, R, or K; X3 is L or I; X4 is T, L, F, K, R, or W; and X5 is W or K.


In certain embodiments, the instant disclosure provides: an antigenic polypeptide comprising an MHC-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, optionally wherein the amino acid sequence of the MHC-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171; and an HSP-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42, optionally wherein the amino acid sequence of the HSP-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42.


In certain embodiments, the C-terminus of the MHC-binding peptide is linked (either directly or indirectly) to the N-terminus of the HSP-binding peptide. Accordingly, in certain embodiments, the antigenic polypeptide comprises an MHC-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, and an HSP-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42, wherein the C-terminus of the MHC-binding peptide is linked (either directly or indirectly) to the N-terminus of the HSP-binding peptide.


In certain embodiments, the N-terminus of the MHC-binding peptide is linked (either directly or indirectly) to the C-terminus of the HSP-binding peptide. Accordingly, in certain embodiments, the antigenic polypeptide comprises an MHC-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, and an HSP-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42, wherein the N-terminus of the MHC-binding peptide is linked (either directly or indirectly) to the C-terminus of the HSP-binding peptide.


In certain embodiments, the MHC-binding peptide is 8 to 50 amino acids in length, optionally 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.


In certain embodiments, the HSP-binding peptide is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In certain embodiments, the HSP-binding peptide is less than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.


In certain embodiments, the HSP-binding peptide is linked to the MHC-binding peptide via a chemical linker. Any chemical linkers can be employed to link the HSP-binding peptide and the MHC-binding peptide. Exemplary chemical linkers include moieties generated from chemical crosslinking (see, e.g., Wong, 1991, Chemistry of Protein Conjugation and Cross-Linking, CRC Press, incorporated herein by reference in its entirety), UV crosslinking, and click chemistry reactions (see, e.g., U.S. Patent Publication 20130266512, which is incorporated by reference herein in its entirety).


In certain embodiments, the HSP-binding peptide is linked to the MHC-binding peptide via a peptide linker (e.g., a peptide linker as disclosed herein). In certain embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 43 or FR. In certain embodiments, the amino acid sequence of the peptide linker consists of the amino acid sequence of SEQ ID NO: 43 or FR.


In certain embodiments, the C-terminus of the MHC-binding peptide is linked by the peptide linker of SEQ ID NO: 43 or FR to the N-terminus of the HSP-binding peptide. Accordingly, in certain embodiments, the antigenic polypeptide comprises from N-terminus to C-terminus: an MHC-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171; the peptide linker of SEQ ID NO: 43 or FR; and an HSP-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42. In certain embodiments, the amino acid sequence of the MHC-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, and the amino acid sequence of the HSP-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42.


In certain embodiments, the antigenic polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1372-3919, 3997-4148, and 4172-4217. In certain embodiments, the amino acid sequence of the antigenic polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1372-3919, 3997-4148, and 4172-4217. In certain embodiments, the antigenic polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1372-3919, 3997-4148, and 4172-4217.


In certain embodiments, the N-terminus of the MHC-binding peptide is linked by the peptide linker of SEQ ID NO: 43 or FR to the C-terminus of the HSP-binding peptide. Accordingly, in certain embodiments the antigenic polypeptide comprises from N-terminus to C-terminus: an HSP-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42; the peptide linker of SEQ ID NO: 43 or FR; and an MHC-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171. In certain embodiments, the amino acid sequence of the MHC-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, and the amino acid sequence of the HSP-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42.


In certain embodiments, the antigenic polypeptide comprises an MHC-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, and wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-97. In certain embodiments, the amino acid sequence of the MHC-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171.


In certain embodiments, the antigenic polypeptide comprises an MHC-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, and wherein the C-terminus of the MHC-binding peptide is linked to the N-terminus of an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-67. In certain embodiments, the amino acid sequence of the MHC-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171.


In certain embodiments, the antigenic peptides disclosed herein are 8 to 100 amino acids, (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids) in length. In certain embodiments, an antigenic peptide is 8 to 50 amino acids in length.


In certain embodiments, the antigenic peptides disclosed herein are less than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length.


In certain embodiments, the amino acid sequence of the antigenic polypeptides disclosed herein does not comprise more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 contiguous amino acids of a protein (e.g., a naturally occurring protein) that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 98-1371, 3921-3996, and 4149-4171.


The antigenic polypeptide disclosed herein can comprise one or more MHC-binding peptides. In certain embodiments, the antigenic peptide comprises one MHC-binding peptides. In certain embodiments, the antigenic polypeptide comprises two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) MHC-binding peptides. The two or more MHC-binding peptides can be linked via a chemical linker or a peptide linker, wherein the peptide linker optionally comprises an amino acid sequence that can be recognized and/or cleaved by a protease.


In certain embodiments, the instant disclosure provides a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-3919 and 3921-4217. In certain embodiments, the polypeptide is 8 to 100 amino acids, (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids) in length. In certain embodiments, the polypeptide peptide is 8 to 50 amino acids in length. In certain embodiments, the amino acid sequence of the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-3919 and 3921-4217. In certain embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-3919 and 3921-4217.


The skilled worker will appreciate that the antigenic polypeptides disclosed herein also encompass derivatives of antigenic polypeptides that are modified during or after synthesis. Such modifications include, but are not limited to: glycosylation, acetylation, methylation, phosphorylation (e.g., phosphorylation of Tyr, Ser, Thr, Arg, Lys, or His on a side chain hydroxyl or amine), formylation, or amidation (e.g., amidation of a C-terminal carboxyl group); derivatization using reactive chemical groups (e.g., derivatization of: free NH2, COOH, or OH groups); specific chemical cleavage (e.g., by cyanogen bromide, hydroxylamine, BNPS-Skatole, acid, NaBH4, or alkali hydrolysis); enzymatic cleavage (e.g., by trypsin, chymotrypsin, papain, V8 protease; oxidation; reduction; etc. Methods for effecting the foregoing modification to antigenic polypeptides are well known in the art.


In certain embodiments, the antigenic polypeptide comprises one or more modified amino acid residues (e.g., in the MHC-binding peptide portion of the antigenic polypeptide). In certain embodiments, the antigenic polypeptide comprises a phosphorylated residue (e.g., a Tyr, Ser, Thr, Arg, Lys, or His that has been phosphorylated on a side chain hydroxyl or amine). In certain embodiments, the antigenic polypeptide comprises a phosphomimetic residue (e.g., a mimetic of a Tyr, Ser, Thr, Arg, Lys, or His amino acid that has been phosphorylated on a side chain hydroxyl or amine). Non-limiting examples of phosphomimetic groups include O-boranophospho, borono, O-dithiophospho, phosphoramide, H-phosphonate, alkylphosphonate, phosphorothioate, phosphodithioate and phosphorofluoridate, any of which may be derivatized on Tyr, Thr, Ser, Arg, Lys, or His residues. In certain embodiments, an Asp or Glu residue is used as a phosphomimetic in place of a phospho-Tyr, phospho-Thr, phospho-Ser, phospho-Arg, phospho-Lys and/or phospho-His residue in a peptide. In certain embodiments, the phosphomimetic residue is a non-hydrolyzable analogue of a phosphorylated residue. Accordingly, in certain embodiments, the antigenic polypeptide comprises a phosphopeptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, wherein a phosphorylated amino acid residue of the phosphopeptide is replaced by a non-hydrolyzable mimetic of the phosphorylated amino acid residue.


The skilled worker will further appreciate that, in certain embodiments, the antigenic polypeptides disclosed herein can comprise one or more natural and/or non-natural amino acids (e.g., D-amino acids), and amino acid analogues and derivatives (e.g., disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine). In certain embodiments, the antigenic polypeptides disclosed herein comprise one or more retro-inverso peptides. A “retro-inverso peptide” refers to a peptide with a reversal of the peptide sequence in two or more positions and inversion of the stereochemistry from L to D configuration in chiral amino acids. Thus, a retro-inverso peptide has reversed termini, reversed direction of peptide bonds, and reversed peptide sequence from N-to-C-terminus, while approximately maintaining the topology of the side chains as in the native peptide sequence. Synthesis of retro-inverso peptide analogues are described in Bonelli, F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A and Viscomi, G. C, J. Chem. Soc. Perkin Trans. 1:697-701 (1985); and U.S. Pat. No. 6,261,569, which are incorporated herein in their entirety by reference.


6.2.1 Production of Antigenic Polypeptides by Chemical Synthesis

Antigenic polypeptides disclosed herein can be synthesized by standard chemical methods including the use of a peptide synthesizer. Conventional peptide synthesis or other synthetic protocols well known in the art can be used.


In certain embodiments, the polypeptide disclosed herein consists of amino acid residues (natural or non-natural) linked by peptide bonds. Such polypeptides can be synthesized, for example, by solid-phase peptide synthesis using procedures similar to those described by Merrifield, 1963, J. Am. Chem. Soc., 85:2149, incorporated herein by reference in its entirety. During synthesis, N-α-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to an insoluble polymeric support i.e., polystyrene beads. The polypeptides are synthesized by linking an amino group of an N-α-deprotected amino acid to an α-carboxyl group of an N-α-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide or 2-(6-Chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation. The most commonly used N-α-protecting groups include Boc which is acid labile and Fmoc which is base labile. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art (See, Atherton, et al., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag, each of which is incorporated herein by reference in its entirety).


In addition, analogs and derivatives of polypeptides can be chemically synthesized as described supra. If desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the peptide sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, designer amino acids such as β-methyl amino acids, C-α-methyl amino acids, and N-α-methyl amino acids.


Polypeptides phosphorylated on the side chains of Tyr, Ser, Thr, Arg, Lys, and His can be synthesized in Fmoc solid phase synthesis using the appropriate side chain protected Fmoc-phospho amino acid. In this way, polypeptides with a combination of phosphorylated and non-phosphorylated Tyr, Ser, Thr, Arg, Lys, and His residues can be synthesized. For example, the method of Staerkaer et al can be applied (1991, Tetrahedron Letters 32: 5389-5392). Other procedures (some for specific amino acids) are detailed in De Bont et al. (1987, Trav. Chim Pays Bas 106: 641, 642), Bannwarth and Trezeciak (1987, Helv. Chim. Acta 70: 175-186), Perich and Johns (1988, Tetrahedron Letters 29: 2369-2372), Kitas et al. (1990, J. Org. Chem. 55:4181-4187), Valerio et al. (1989, Int. J. Peptide Protein Res. 33:428-438), Perich et al. (1991, Tetrahedron Letters 32:4033-4034), Pennington (1994, Meth. Molec. Biol. 35:195-2), and Perich (1997, Methods Enzymol. 289:245-266, each of which is incorporated herein by reference in its entirety).


A phosphorylated polypeptide can also be produced by first culturing a cell transformed with a nucleic acid that encodes the amino acid sequence of the polypeptide. After producing such a polypeptide by cell culture, the hydroxyl groups of the appropriate amino acid are substituted by phosphate groups using organic synthesis or enzymatic methods with phosphorylation enzymes. For example, in the case of serine-specific phosphorylation, serine kinases can be used.


Phosphopeptide mimetics can also be synthesized, wherein a phosphorylated amino acid residue in a polypeptide is replaced with a phosphomimetic group. Non-limiting examples of phosphomimetic groups include O-boranophospho, borono, O-dithiophospho, phosphoramide, H-phosphonate, alkylphosphonate, phosphorothioate, phosphodithioate and phosphorofluoridate, any of which may be derivatized on Tyr, Thr, Ser, Arg, Lys, or His residues. In certain embodiments, an Asp or Glu residue is used as a phosphomimetic. Asp or Glu residues can also function as phosphomimetic groups, and be used in place of a phospho-Tyr, phospho-Thr, phospho-Ser, phospho-Arg, phospho-Lys and/or phospho-His residue in a peptide.


Purification of the resulting peptide is accomplished using conventional procedures, such as preparative HPLC using reverse-phase, gel permeation, partition and/or ion exchange chromatography. The choice of appropriate matrices and buffers are well known in the art and so are not described in detail herein.


6.2.2 Production of Antigenic Polypeptides Using Recombinant DNA Technology

Polypeptides disclosed herein can also be prepared by recombinant DNA methods known in the art. A nucleic acid sequence encoding a polypeptide can be obtained by back translation of the amino acid sequence and synthesized by standard chemical methods, such as the use of an oligonucleotide synthesizer. Alternatively, coding information for polypeptides can be obtained from DNA templates using specifically designed oligonucleotide primers and PCR methodologies. Variations and fragments of the polypeptides can be made by substitutions, insertions or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, DNA sequences which encode the same or a variant of a polypeptide may be used in the practice of the present invention. These include, but are not limited to, nucleotide sequences which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent or conservative change. The nucleic acid encoding a polypeptide can be inserted into an expression vector for propagation and expression in host cells.


As the coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Soc. 103:3185 (1981) (incorporated herein by reference in its entirety), modification can be made simply by substituting the appropriate base(s) for those encoding the native peptide sequence. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired peptide or fusion protein. A number of such vectors and suitable host systems are now available. For expression of the peptide or fusion proteins, the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host.


An expression construct refers to a nucleotide sequence encoding a polypeptide operably linked with one or more regulatory regions which enables expression of the peptide in an appropriate host cell. “Operably-linked” refers to an association in which the regulatory regions and the peptide sequence to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation.


The regulatory regions necessary for transcription of the peptide can be provided by the expression vector. A translation initiation codon (ATG) may also be provided if the peptide gene sequence lacking its cognate initiation codon is to be expressed. In a compatible host-construct system, cellular transcriptional factors, such as RNA polymerase, will bind to the regulatory regions on the expression construct to effect transcription of the peptide sequence in the host organism. The precise nature of the regulatory regions needed for gene expression may vary from host cell to host cell. Generally, a promoter is required which is capable of binding RNA polymerase and promoting the transcription of an operably-associated nucleic acid sequence. Such regulatory regions may include those 5′ non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like. The non-coding region 3′ to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites.


In order to attach DNA sequences with regulatory functions, such as promoters, to the peptide gene sequence or to insert the peptide gene sequence into the cloning site of a vector, linkers or adapters providing the appropriate compatible restriction sites may be ligated to the ends of the cDNAs by techniques well known in the art (Wu et al., 1987, Methods in Enzymol 152:343-349, incorporated herein by reference in its entirety). Cleavage with a restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation. Alternatively, a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA by use of PCR with primers containing the desired restriction enzyme site.


An expression construct comprising a polypeptide coding sequence operably linked with regulatory regions can be directly introduced into appropriate host cells for expression and production of the peptide without further cloning. The expression constructs can also contain DNA sequences that facilitate integration of the DNA sequence into the genome of the host cell, e.g., via homologous recombination. In this instance, it is not necessary to use an expression vector comprising a replication origin suitable for appropriate host cells in order to propagate and express the peptide in the host cells.


A variety of expression vectors may be used including plasmids, cosmids, phage, phagemids or modified viruses. Typically, such expression vectors comprise a functional origin of replication for propagation of the vector in an appropriate host cell, one or more restriction endonuclease sites for insertion of the peptide gene sequence, and one or more selection markers. Expression vectors may be constructed to carry nucleotide sequences for one or more of the polypeptides disclosed herein. The expression vector must be used with a compatible host cell which may be derived from a prokaryotic or eukaryotic organism including but not limited to bacteria, yeasts, insects, mammals and humans. Such host cells can be transformed to express one or more polypeptides disclosed herein, such as by transformation of the host cell with a single expression vector containing a plurality of nucleotide sequences encoding any of the polypeptides disclosed herein, or by transformation of the host cell with multiple expression vectors encoding different polypeptides disclosed herein.


In bacterial systems, a number of expression vectors may be advantageously selected to produce polypeptides. For example, when a large quantity of such a protein is to be produced, such as for the generation of pharmaceutical compositions, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2, 1791, incorporated herein by reference in its entirety), in which the peptide coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13, 3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem 264, 5503-5509, each of which is incorporated herein by reference in its entirety); and the like. pGEX vectors may also be used to express these peptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the polypeptide can be released from the GST moiety.


Alternatively, for long term, high yield production of properly processed peptide complexes, stable expression in mammalian cells is preferred. Cell lines that stably express peptide complexes may be engineered by using a vector that contains a selectable marker. By way of example, following the introduction of the expression constructs, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the expression construct confers resistance to the selection and optimally allows cells to stably integrate the expression construct into their chromosomes and to grow in culture and to be expanded into cell lines. Such cells can be cultured for a long period of time while the peptide is expressed continuously.


The recombinant cells may be cultured under standard conditions of temperature, incubation time, optical density and media composition. However, conditions for growth of recombinant cells may be different from those for expression of the polypeptides. Modified culture conditions and media may also be used to enhance production of the peptides. For example, recombinant cells containing peptides with their cognate promoters may be exposed to heat or other environmental stress, or chemical stress. Any techniques known in the art may be applied to establish the optimal conditions for producing peptide complexes.


In one embodiment disclosed herein, a codon encoding methionine is added at the 5′ end of the nucleotide sequence encoding a polypeptide to provide a signal for initiation of translation of the peptide. This methionine may remain attached to the polypeptide, or the methionine may be removed by the addition of an enzyme or enzymes that can catalyze the cleavage of methionine from the peptide. For example, in both prokaryotes and eukaryotes, N-terminal methionine is removed by a methionine aminopeptidase (MAP) (Tsunasawa et al., 1985, J. Biol. Chem. 260, 5382-5391, incorporated herein by reference in its entirety). Methionine aminopeptidases have been isolated and cloned from several organisms, including E. coli, yeast, and rat.


The peptide may be recovered from the bacterial, mammalian, or other host cell types, or from the culture medium, by known methods (see, for example, Current Protocols in Immunology, vol. 2, chapter 8, Coligan et al. (ed.), John Wiley & Sons, Inc.; Pathogenic and Clinical Microbiology: A Laboratory Manual by Rowland et al., Little Brown & Co., June 1994, incorporated herein by reference in its entirety).


Both of the foregoing methods can be used for synthesizing a polypeptide disclosed herein. For example, a peptide comprising the amino acid sequence of the HSP-binding peptide can be synthesized chemically, and joined to an antigenic peptide, optionally produced by recombinant DNA technology, via a peptide bond.


Included within the scope disclosed herein are derivatives or analogs of the polypeptides disclosed herein that are modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation (e.g., of the C-terminal carboxyl group), or derivatization by known protecting/blocking groups, or proteolytic cleavage. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, reagents useful for protection or modification of free NH2— groups, free COOH— groups, OH— groups, side groups of Trp-, Tyr-, Phe-, His-, Arg-, or Lys-; specific chemical cleavage by cyanogen bromide, hydroxylamine, BNPS-Skatole, acid, or alkali hydrolysis; enzymatic cleavage by trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.


6.3 Compositions Comprising Antigenic Polypeptides

In another aspect, the instant disclosure provides a composition (e.g., a pharmaceutical composition, a vaccine, or a unit dosage form thereof) comprising one or more antigenic polypeptide as disclosed herein. In certain embodiments, the composition comprises a plurality of the antigenic polypeptides disclosed herein. For example, in certain embodiments, the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 different antigenic polypeptides as disclosed herein.


6.3.1 Compositions Comprising Antigenic Polypeptides in Complex with Stress Proteins


In certain embodiments, the instant disclosure provides a composition (e.g., a pharmaceutical composition) comprising one or more antigenic polypeptides as disclosed herein and a purified stress protein. In certain embodiments, at least a portion of the purified stress protein binds to the antigenic polypeptide in the composition. Such compositions are useful as vaccines for the treatment of a cancer.


Stress proteins, which are also referred to interchangeably herein as heat shock proteins (HSPs), useful in the practice of the instant invention can be selected from among any cellular protein that is capable of binding other proteins or peptides and capable of releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) or under acidic conditions. The intracellular concentration of such protein may increase when a cell is exposed to a stressful stimulus. In addition to those heat shock proteins that are induced by stress, the HSP60, HSP70, HSP90, HSP100, sHSPs, and PDI families also include proteins that are related to stress-induced HSPs in sequence similarity, for example, having greater than 35% amino acid identity, but whose expression levels are not altered by stress. Therefore, stress protein or heat shock protein embraces other proteins, mutants, analogs, and variants thereof having at least 35% (e.g., at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%) amino acid identity with members of these families whose expression levels in a cell are enhanced in response to a stressful stimulus. Accordingly, in certain embodiments, the stress protein is a member of the hsp60, hsp70, or hsp90 family of stress proteins (e.g., Hsc70, human Hsc70), or a mutant, analog, or variant thereof. In certain embodiments, the stress protein is selected from the group consisting of hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, a mutant thereof, and combinations of two or more thereof. In certain embodiments, the stress protein is Hsc70 (e.g., human Hsc70). In certain embodiments, the stress protein comprises the amino acid sequence of SEQ ID NO: 3920. In certain embodiments, the amino acid sequence of the stress protein consists of the amino acid sequence of SEQ ID NO: 3920. In certain embodiments, the stress protein is Hsp70 (e.g., human Hsp70). In certain embodiments, the stress protein (e.g., human hsc70) is a recombinant protein.


Amino acid sequences and nucleotide sequences of naturally occurring HSPs are generally available in sequence databases, such as GenBank. For example, Homo sapiens heat shock protein HSP70 (Heat Shock 70 kDa Protein 1A) has the following identifiers HGNC: 5232; Entrez Gene: 3303; Ensembl: ENSG00000204389; OMIM: 140550; UniProtKB: P08107 and NCBI Reference Sequence: NM_005345.5. Computer programs, such as Entrez, can be used to browse the database, and retrieve any amino acid sequence and genetic sequence data of interest by accession number. These databases can also be searched to identify sequences with various degrees of similarities to a query sequence using programs, such as FASTA and BLAST, which rank the similar sequences by alignment scores and statistics. Nucleotide sequences of non-limiting examples of HSPs that can be used for preparation of the HSP peptide-binding fragments disclosed herein are as follows: human Hsp70, Genbank Accession No. NM_005345, Sargent et al., 1989, Proc. Natl. Acad. Sci. U.S.A., 86:1968-1972; human Hsc70: Genbank Accession Nos. P11142, Y00371; human Hsp90, Genbank Accession No. X15183, Yamazaki et al., Nucl. Acids Res. 17:7108; human gp96: Genbank Accession No. X15187, Maki et al., 1990, Proc. Natl. Acad Sci., 87: 5658-5562; human BiP: Genbank Accession No. M19645; Ting et al., 1988, DNA 7: 275-286; human Hsp27, Genbank Accession No. M24743; Hickey et al., 1986, Nucleic Acids Res. 14:4127-45; mouse Hsp70: Genbank Accession No. M35021, Hunt et al., 1990, Gene, 87:199-204; mouse gp96: Genbank Accession No. M16370, Srivastava et al., 1987, Proc. Natl. Acad. Sci., 85:3807-3811; and mouse BiP: Genbank Accession No. U16277, Haas et al., 1988, Proc. Natl. Acad. Sci. U.S.A., 85: 2250-2254 (each of these references is incorporated herein by reference in its entirety).


In addition to the major stress protein families described above, an endoplasmic reticulum resident protein, calreticulin, has also been identified as yet another heat shock protein useful for eliciting an immune response when complexed to antigenic molecules (Basu and Srivastava, 1999, J. Exp. Med. 189:797-202; incorporated herein by reference in its entirety). Other stress proteins that can be used in the invention include grp78 (or BiP), protein disulfide isomerase (PDI), hsp110, and grp170 (Lin et al., 1993, Mol. Biol. Cell, 4:1109-1119; Wang et al., 2001, J. Immunol., 165:490-497, each of which is incorporated herein by reference in its entirety). Many members of these families were found subsequently to be induced in response to other stressful stimuli including nutrient deprivation, metabolic disruption, oxygen radicals, hypoxia and infection with intracellular pathogens (see Welch, May 1993, Scientific American 56-64; Young, 1990, Annu. Rev. Immunol. 8:401-420; Craig, 1993, Science 260:1902-1903; Gething, et al., 1992, Nature 355:33-45; and Lindquist, et al., 1988, Annu. Rev. Genetics 22:631-677, each of which is incorporated herein by reference in its entirety). It is contemplated that HSPs/stress proteins belonging to all of these families can be used in the practice disclosed herein. In certain embodiments, a stress protein encompasses any chaperone protein that facilitates peptide-MHC presentation. Suitable chaperone proteins include, but are not limited to, ER chaperones and tapasin (e.g., human tapasin).


The major stress proteins can accumulate to very high levels in stressed cells, but they occur at low to moderate levels in cells that have not been stressed. For example, the highly inducible mammalian hsp70 is hardly detectable at normal temperatures but becomes one of the most actively synthesized proteins in the cell upon heat shock (Welch, et al., 1985, J. Cell. Biol. 101:1198-1211, incorporated herein by reference in its entirety). In contrast, hsp90 and hsp60 proteins are abundant at normal temperatures in most, but not all, mammalian cells and are further induced by heat (Lai, et al., 1984, Mol. Cell. Biol. 4:2802-10; van Bergen en Henegouwen, et al., 1987, Genes Dev. 1:525-31, each of which is incorporated herein by reference in its entirety).


In various embodiments, nucleotide sequences encoding heat shock protein within a family or variants of a heat shock protein can be identified and obtained by hybridization with a probe comprising nucleotide sequence encoding an HSP under conditions of low to medium stringency. By way of example, procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792). Filters containing DNA are pretreated for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate. Filters are incubated in hybridization mixture for 18-20 h at 40° C., and then washed for 1.5 h at 55° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for signal detection. If necessary, filters are washed for a third time at 65-68° C. before signal detection. Other conditions of low stringency which may be used are well known in the art (e.g., as used for cross-species hybridizations).


Where stress proteins are used, peptide-binding fragments of stress proteins and functionally active derivatives, analogs, and variants thereof can also be used. Accordingly, in certain embodiments, the stress protein is a full-length HSP. In certain embodiments, the stress protein is a polypeptide comprising a domain of an HSP (e.g., a member of the Hsp60, Hsp70, or Hsp90 family, such as Hsc70, particularly human Hsc70), wherein the domain is capable of being noncovalently associated with a peptide (e.g., an HSP-binding peptide as described herein) to form a complex and optionally eliciting an immune response, and wherein the stress protein is not a full-length HSP.


In certain embodiments, the stress protein is a polypeptide that is capable of being noncovalently associated with a peptide (e.g., an HSP-binding peptide as described herein) to form a complex and optionally eliciting an immune response, wherein the stress protein shares a high degree of sequence similarity with a wild-type HSP (e.g., a member of the Hsp60, Hsp70, or Hsp90 family, such as Hsc70, particularly human Hsc70). To determine a region of identity between two amino acid sequences or nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions×100%). In one embodiment, the two sequences are the same length.


The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877 (each of which is incorporated herein by reference in its entirety). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al., 1990, J. Mol. Biol. 215:403-410 (incorporated herein by reference in its entirety). BLAST nucleotide searches can be performed with the NBLAST program, e.g., score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule disclosed herein. BLAST protein searches can be performed with the XBLAST program, e.g., score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule disclosed herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Altschul et al., 1997, supra). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.


In certain embodiments, isolated peptide-binding domains of a stress protein (e.g., Hsp70 or Hsc70) are employed. These peptide-binding domains can be identified by computer modeling of the three-dimensional structure of the peptide-binding site of a stress protein (e.g., Hsp70 and Hsc70). See for example, the peptide-binding fragments of HSPs disclosed in United States patent publication US 2001/0034042 (incorporated herein by reference in its entirety).


In certain embodiments, the stress protein is a mutated stress protein which has an affinity for a target polypeptide that is greater than a native stress protein. Such mutated stress proteins can be useful when the target polypeptide is phosphorylated or is a phosphopeptide mimetic (such as non-hydrolyzable analogs) or has some other post-translational modification.


The stress proteins can be prepared by purification from tissues, or by recombinant DNA techniques. HSPs can be purified from tissues in the presence of ATP or under acidic conditions (pH 1 to pH 6.9), for subsequent in vitro complexing to one or more polypeptides. See Peng, et al., 1997, J. Immunol. Methods, 204:13-21; Li and Srivastava, 1993, EMBO J. 12:3143-3151 (each of these references is incorporated herein by reference in its entirety). “Purified” stress proteins are substantially free of materials that are associated with the proteins in a cell, in a cell extract, in a cell culture medium, or in an individual. In certain embodiments, the stress protein purified from a tissue is a mixture of different HSPs, for example, hsp70 and hsc70.


Using the defined amino acid or cDNA sequences of a given HSP or a peptide-binding domain thereof, one can make a genetic construct which is transfected into and expressed in a host cell. The recombinant host cells may contain one or more copies of a nucleic acid sequence comprising a sequence that encodes an HSP or a peptide-binding fragment, operably linked with regulatory region(s) that drives the expression of the HSP nucleic acid sequence in the host cell. Recombinant DNA techniques can be readily utilized to generate recombinant HSP genes or fragments of HSP genes, and standard techniques can be used to express such HSP gene fragments. Any nucleic acid sequence encoding an HSP peptide-binding domain, including cDNA and genomic DNA, can be used to prepare the HSPs or peptide-binding fragments disclosed herein. The nucleic acid sequence can be wild-type or a codon-optimized variant that encodes the same amino acid sequence. An HSP gene fragment containing the peptide-binding domain can be inserted into an appropriate cloning vector and introduced into host cells so that many copies of the gene sequence are generated. A large number of vector-host systems known in the art may be used such as, but not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322, pUC plasmid derivatives, the Bluescript vectors (Stratagene) or the pET series of vectors (Novagen). Any technique for mutagenesis known in the art can be used to modify individual nucleotides in a DNA sequence, for purpose of making amino acid substitution(s) in the expressed peptide sequence, or for creating/deleting restriction sites to facilitate further manipulations.


The stress proteins may be expressed as fusion proteins to facilitate recovery and purification from the cells in which they are expressed. For example, the stress proteins may contain a signal sequence leader peptide to direct its translocation across the endoplasmic reticulum membrane for secretion into culture medium. Further, the stress protein may contain an affinity label fused to any portion of the protein not involved in binding to a target polypeptide, for example, the carboxyl terminus. The affinity label can be used to facilitate purification of the protein, by binding to an affinity partner molecule. A variety of affinity labels known in the art may be used, non-limiting examples of which include the immunoglobulin constant regions, polyhistidine sequence (Petty, 1996, Metal-chelate affinity chromatography, in Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, incorporated herein by reference in its entirety), glutathione S-transferase (GST; Smith, 1993, Methods Mol. Cell Bio. 4:220-229, incorporated herein by reference in its entirety), the E. coli maltose binding protein (Guan et al., 1987, Gene 67:21-30, incorporated herein by reference in its entirety), and various cellulose binding domains (U.S. Pat. Nos. 5,496,934; 5,202,247; 5,137,819; Tomme et al., 1994, Protein Eng. 7:117-123, each of which is incorporated herein by reference in its entirety).


Such recombinant stress proteins can be assayed for peptide binding activity (see, e.g., Klappa et al., 1998, EMBO J., 17:927-935, incorporated herein by reference in its entirety) for their ability to elicit an immune response. In certain embodiments, the recombinant stress protein produced in the host cell is of the same species as the intended recipient of the immunogenic composition (e.g., human).


The stress protein may be bound to the polypeptide(s) non-covalently or covalently. In certain embodiments, the stress protein is non-covalently bound to the polypeptide. Methods of preparing such complexes are set forth infra.


The molar ratio of total polypeptide(s) to total stress protein(s) can be any ratio from about 0.01:1 to about 100:1, including but not limited to about 0.01:1, 0.02:1, 0.05:1. 0.1:1. 0.2:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 49:1, up to 100:1. In certain embodiments, the composition comprises a plurality of complexes each comprising a polypeptide disclosed herein and a stress protein, wherein the molar ratio of the polypeptide to the stress protein in each complex is at least about 1:1 (e.g., about 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 49:1, up to 100:1).


In certain embodiments, the molar ratio of total polypeptide(s) to total stress protein(s) is about 0.5:1 to 5:1. In certain embodiments, the molar ratio of total polypeptide(s) to total stress protein(s) is about 1:1 to 2:1. In certain embodiments, the molar ratio of total polypeptide(s) to total stress protein(s) is about 1:1, 1.25:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1. Such ratios, particularly the ratios close to 1:1, are advantageous in that the composition does not comprise a great excess of free peptide(s) that is not bound to a stress protein. Since many antigenic peptides comprising MHC-binding peptides tend to comprise hydrophobic regions, an excess amount of free peptide(s) may tend to aggregate during preparation and storage of the composition. Substantial complexation with a stress protein at a molar ratio of total polypeptide(s) to total stress protein(s) close to 1:1 (e.g., 1:1, 1.25:1, 1.5:1, or 2:1) is enabled by a high binding affinity of the polypeptide to the stress protein. Accordingly, in certain embodiments, the polypeptide binds to an HSP (e.g., Hsc70, Hsp70, Hsp90, Hsp110, Grp170, Gp96, or Calreticulin) with a Kd lower than 10−3 M, 10−4 M, 10−5 M, 10−6 M, 10−7 M, 10−8 M, or 10−9 M. In certain embodiments, the polypeptide binds to Hsc70 (e.g., human Hsc70) with a Kd of 10−3 M, 10−4 M, 10−5 M, 10−6 M, 10−7 M, 10−8 M, 10−9 M, or lower.


In certain embodiments, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the stress protein binds to the polypeptide in the composition. In certain embodiments, substantially all of the stress protein binds to the polypeptide in the composition.


Any number of different polypeptides can be included in a single composition as disclosed herein. In certain embodiments, the compositions comprise no more than 100 different polypeptides, e.g., 2-50, 2-30, 2-20, 5-20, 5-15, 5-10, or 10-15 different polypeptides.


In certain embodiments, each of the antigenic polypeptides comprises the same HSP-binding peptide and a different antigenic peptide. In certain embodiments, the composition comprises a single stress protein, wherein the stress protein is capable of binding to the HSP-binding peptide.


Pharmaceutical compositions comprising the complexes of stress proteins and antigenic polypeptides disclosed herein can be formulated to contain one or more pharmaceutically acceptable carriers or excipients including bulking agents, stabilizing agents, buffering agents, sodium chloride, calcium salts, surfactants, antioxidants, chelating agents, other excipients, and combinations thereof.


Bulking agents are preferred in the preparation of lyophilized formulations of the composition. Such bulking agents form the crystalline portion of the lyophilized product and may be selected from the group consisting of mannitol, glycine, alanine, and hydroxyethyl starch (HES).


Stabilizing agents may be selected from the group consisting of sucrose, trehalose, raffinose, and arginine. These agents are preferably present in amounts between 1-4%. Sodium chloride can be included in the present formulations preferably in an amount of 100-300 mM, or if used without the aforementioned bulking agents, can be included in the formulations in an amount of between 300-500 mM NaCl. Calcium salts include calcium chloride, calcium gluconate, calcium glubionate, or calcium gluceptate.


Buffering agents can be any physiologically acceptable chemical entity or combination of chemical entities which have a capacity to act as buffers, including but not limited to histidine, potassium phosphate, TRIS [tris-(hydroxymethyl)-aminomethane], BIS-Tris Propane (1,3-bis-[tris-(hydroxymethyl)methylamino]-propane), PIPES [piperazine-N,N′-bis-(2-ethanesulfonic acid)], MOPS [3-(N-morpholino)ethanesulfonic acid], HEPES (N-2-hydroxyethyl-piperazine-N′-2-ethanesulfonic acid), MES [2-(N-morpholino)ethanesulfonic acid], and ACES (N-2-acetamido-2-aminoethanesulfonic acid). Typically, the buffering agent is included in a concentration of 10-50 mM. Specific examples of base buffers include (i) PBS; (ii) 10 mM KPO4, 150 mM NaCl; (iii) 10 mM HEPES, 150 mM NaCl; (iv) 10 mM imidazole, 150 mM NaCl; and (v) 20 mM sodium citrate. Excipients that can be used include (i) glycerol (10%, 20%); (ii) Tween 50 (0.05%, 0.005%); (iii) 9% sucrose; (iv) 20% sorbitol; (v) 10 mM lysine; or (vi) 0.01 mM dextran sulfate.


Surfactants, if present, are preferably in a concentration of 0.1% or less, and may be chosen from the group including but not limited to polysorbate 20, polysorbate 80, pluronic polyols, and BRIJ 35 (polyoxyethylene 23 laurel ether). Antioxidants, if used, must be compatible for use with a pharmaceutical preparation, and are preferably water soluble. Suitable antioxidants include homocysteine, glutathione, lipoic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), methionine, sodium thiosulfate, platinum, glycine-glycine-histidine (tripeptide), and butylatedhydroxytoluene (BHT). Chelating agents should preferably bind metals such as copper and iron with greater affinity than calcium, if a calcium salt is being used in the composition. An exemplary chelator is deferoxamine.


Many formulations known in the art can be used. For example, U.S. Pat. No. 5,763,401 describes a therapeutic formulation, comprising 15-60 mM sucrose, up to 50 mM NaCl, up to 5 mM calcium chloride, 65-400 mM glycine, and up to 50 mM histidine. In some embodiments, the therapeutic formulation is a solution of 9% sucrose in potassium phosphate buffer.


U.S. Pat. No. 5,733,873 (incorporated herein by reference in its entirety) discloses formulations which include between 0.01-1 mg/ml of a surfactant. This patent discloses formulations having the following ranges of excipients: polysorbate 20 or 80 in an amount of at least 0.01 mg/ml, preferably 0.02-1.0 mg/ml; at least 0.1 M NaCl; at least 0.5 mM calcium salt; and at least 1 mM histidine. More particularly, the following specific formulations are also disclosed: (1) 14.7-50-65 mM histidine, 0.31-0.6 M NaCl, 4 mM calcium chloride, 0.001-0.02-0.025% polysorbate 80, with or without 0.1% PEG 4000 or 19.9 mM sucrose; and (2) 20 mg/ml mannitol, 2.67 mg/ml histidine, 18 mg/ml NaCl, 3.7 mM calcium chloride, and 0.23 mg/ml polysorbate 80.


The use of low or high concentrations of sodium chloride has been described, for example U.S. Pat. No. 4,877,608 (incorporated herein by reference in its entirety) teaches formulations with relatively low concentrations of sodium chloride, such as formulations comprising 0.5 mM-15 mM NaCl, 5 mM calcium chloride, 0.2 mM-5 mM histidine, 0.01-10 mM lysine hydrochloride and up to 10% maltose, 10% sucrose, or 5% mannitol.


U.S. Pat. No. 5,605,884 (incorporated herein by reference in its entirety) teaches the use of formulations with relatively high concentrations of sodium chloride. These formulations include 0.35 M-1.2 M NaCl, 1.5-40 mM calcium chloride, 1 mM-50 mM histidine, and up to 10% sugar such as mannitol, sucrose, or maltose. A formulation comprising 0.45 M NaCl, 2.3 mM calcium chloride, and 1.4 mM histidine is exemplified.


International Patent Application WO 96/22107 (incorporated herein by reference in its entirety) describes formulations which include the sugar trehalose, for example formulations comprising: (1) 0.1 M NaCl, 15 mM calcium chloride, 15 mM histidine, and 1.27 M (48%) trehalose; or (2) 0.011% calcium chloride, 0.12% histidine, 0.002% TRIS, 0.002% Tween 80, 0.004% PEG 3350, 7.5% trehalose; and either 0.13% or 1.03% NaCl.


U.S. Pat. No. 5,328,694 (incorporated herein by reference in its entirety) describes a formulation which includes 100-650 mM disaccharide and 100 mM-1.0 M amino acid, for example (1) 0.9 M sucrose, 0.25 M glycine, 0.25 M lysine, and 3 mM calcium chloride; and (2) 0.7 M sucrose, 0.5 M glycine, and 5 mM calcium chloride. Pharmaceutical compositions can be optionally prepared as lyophilized product, which may then be formulated for oral administration or reconstituted to a liquid form for parenteral administration.


In certain embodiments, the composition stimulates a T-cell response against a cell expressing or displaying a polypeptide comprising one or more of the MHC-binding peptides in a subject to whom the composition is administered. The cell expressing the polypeptide may be a cell comprising a polynucleotide encoding the polypeptide, wherein the polynucleotide is in the genome of the cell, in an episomal vector, or in the genome of a virus that has infected the cell. The cell displaying the polypeptide may not comprise a polynucleotide encoding the polypeptide, and may be produced by contacting the cell with the polypeptide or a derivative thereof.


In certain embodiments, the composition induces in vitro activation of T cells in peripheral blood mononuclear cells (PBMCs) isolated from a subject. The in vitro activation of T cells includes, without limitation, in vitro proliferation of T cells, production of cytokines (e.g., IFNγ) from T cells, and increased surface expression of activation markers (e.g., CD25, CD45RO) on T cells.


6.3.2 Preparation of Complexes of Antigenic Polypeptides and Stress Proteins

In another aspect, the instant disclosure provides a method of making complexes of antigenic polypeptides and stress proteins (e.g., for the purposes of making a vaccine), the method comprising mixing one or more antigenic polypeptides as disclosed herein with a purified stress protein in vitro under suitable conditions such that the purified stress protein binds to at least one of the antigenic polypeptides. The method is also referred to as a complexing reaction herein. In certain embodiments, two or more purified stress proteins are employed, wherein each purified stress protein binds to at least one of the antigenic polypeptides. In certain embodiments, at least a portion of the purified stress protein binds to the antigenic polypeptide in the composition.


The stress protein may be bound to the polypeptide non-covalently or covalently. In certain embodiments, the stress protein is non-covalently bound to the polypeptide. In various embodiments, the complexes formed in vitro are optionally purified. Purified complexes of stress proteins and polypeptides are substantially free of materials that are associated with such complexes in a cell, or in a cell extract. Where purified stress proteins and purified polypeptides are used in an in vitro complexing reaction, the term “purified complex(es)” does not exclude a composition that also comprises free stress proteins and conjugates or peptides not in complexes.


Any stress proteins described supra may be employed in the method disclosed herein. In certain embodiments, the stress protein is selected from the group consisting of Hsc70, Hsp70, Hsp90, Hsp110, Grp170, Gp96, Calreticulin, a mutant thereof, and combinations of two or more thereof. In one embodiment, the stress protein is an Hsc70, e.g., a human Hsc70. In another embodiment, the stress protein is an Hsp70, e.g., a human Hsp70. In certain embodiments, the stress protein (e.g., human Hsc70 or human Hsp70) is a recombinant protein.


Prior to complexing, HSPs can be pretreated with ATP or exposed to acidic conditions to remove any peptides that may be non-covalently associated with the HSP of interest. Acidic conditions are any pH levels below pH 7, including the ranges pH 1-pH 2, pH 2-pH 3, pH 3-pH 4, pH 4-pH 5, pH 5-pH 6, and pH 6-pH 6.9. When the ATP procedure is used, excess ATP is removed from the preparation by the addition of apyranase as described by Levy, et al., 1991, Cell 67:265-274 (incorporated herein by reference in its entirety). When acidic conditions are used, the buffer is readjusted to neutral pH by the addition of pH modifying reagents.


In certain embodiments, prior to complexation with purified stress proteins, the polypeptides may be reconstituted from powder in 100% DMSO. Equimolar amounts of the peptides may then be pooled in a solution of 75% DMSO diluted in sterile water.


In certain embodiments, prior to complexation with purified stress proteins, the polypeptides may be reconstituted in neutral water.


In certain embodiments, prior to complexation with purified stress proteins, the polypeptides may be reconstituted in acidic water containing HCl.


In certain embodiments, prior to complexation with purified stress proteins, the polypeptides may be reconstituted in basic water containing NaOH.


In certain embodiments, prior to complexation with purified stress proteins, the solubility of each polypeptide in water may be tested. If a polypeptide is soluble in neutral water, neutral water may be used as a solvent for the polypeptide. If the polypeptide is not soluble in neutral water, solubility in acidic water containing HCl, or another acid, e.g., acetic acid, phosphoric acid, or sulfuric acid may be tested. If the polypeptide is soluble in acidic water containing HCl (or another acid), acidic water containing HCl (or another acid) may be used as the solvent for the polypeptide. If the polypeptide is not soluble in acidic water containing HCl (or another acid), solubility in basic water containing NaOH may be tested. If the polypeptide is soluble in basic water containing NaOH, basic water containing NaOH may be used as the solvent for the polypeptide. If the polypeptide is not soluble in basic water containing NaOH, the polypeptide may be dissolved in DMSO. If the polypeptide is not soluble in DMSO the polypeptide may be excluded. The dissolved polypeptides may then be mixed to make a pool of polypeptides. The dissolved polypeptides may be mixed at equal volume. The dissolved polypeptides may be mixed in equimolar amounts.


The molar ratio of total polypeptide(s) to total stress protein(s) can be any ratio from 0.01:1 to 100:1, including but not limited to 0.01:1, 0.02:1, 0.05:1. 0.1:1. 0.2:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 49:1, up to 100:1. In certain embodiments, the composition to be prepared comprises a plurality of complexes each comprising a polypeptide disclosed herein and a stress protein, and the complexing reaction comprises mixing the polypeptides with the stress proteins, wherein the molar ratio of the polypeptide to the stress protein in each complex is at least 1:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 49:1, up to 100:1).


In certain embodiments, the molar ratio of total polypeptide(s) to total stress protein(s) is about 0.5:1 to 5:1. In certain embodiments, the molar ratio of total polypeptide(s) to total stress protein(s) is about 1:1 to 2:1. In certain embodiments, the molar ratio of total polypeptide(s) to total stress protein(s) is about 1:1, 1.25:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1. Such ratios, particularly the ratios close to 1:1, are advantageous in that the composition does not comprise a great excess of free peptide(s) that is not bound to a stress protein. Since many antigenic peptides comprising MHC-binding peptides tend to comprise hydrophobic regions, an excess amount of free peptide(s) may tend to aggregate during preparation and storage of the composition. Substantial complexation with a stress protein at a molar ratio of total polypeptide(s) to total stress protein(s) close to 1:1 (e.g., 1:1, 1.25:1, 1.5:1, or 2:1) is enabled by a high binding affinity of the polypeptide to the stress protein. Accordingly, in certain embodiments, the polypeptide used in the complexing reaction binds to an HSP (e.g., Hsc70, Hsp70, Hsp90, Hsp110, Grp170, Gp96, or Calreticulin) with a Kd lower than 10−3 M, 10−4 M, 10−5 M, 10−6 M, 10−7 M, 10−8 M, or 10−9 M. In certain embodiments, the polypeptide binds to Hsc70 (e.g., human Hsc70) with a Kd of 10−3 M, 10−4 M, 10−5 M, 10−6 M, 10−7 M, 10−8 M, 10−9 M, or lower.


The method disclosed herein can be used to prepare a composition (e.g., a pharmaceutical composition) in bulk (e.g., greater than or equal to 30 mg, 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, or 1 g of total peptide and protein). The prepared composition can then be transferred to single-use or multi-use containers, or apportioned to unit dosage forms. Alternatively, the method disclosed herein can be used to prepare a composition (e.g., a pharmaceutical composition) in a small amount (e.g., less than or equal to 300 μg, 1 mg, 3 mg, 10 mg, 30 mg, or 100 mg of total peptide and protein). In certain embodiments, the composition is prepared for single use, optionally in a unit dosage form.


In certain embodiments, the total amount of the polypeptide(s) and stress protein in the composition is about 10 μg to 600 μg (e.g., about 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, or 500 g, optionally about 120 μg, 240 μg, or 480 μg). In certain embodiments, the total amount of the polypeptide(s) and stress protein in the composition is about 300 μg. Amounts of the stress protein(s) and polypeptide(s) in a unit dosage form are disclosed infra.


An exemplary protocol for noncovalent complexing of a population of polypeptides to a stress protein in vitro is provided herein. The population of polypeptides can comprise a mixture of the different polypeptide species disclosed herein. Then, the mixture is incubated with the purified and/or pretreated stress protein for from 15 minutes to 3 hours (e.g., 1 hour) at from 4° to 50° C. (e.g., 37° C.) in a suitable binding buffer, such as phosphate buffered saline pH 7.4 optionally supplemented with 0.01% Polysorbate 20; a buffer comprising 9% sucrose in potassium phosphate buffer; a buffer comprising 2.7 mM Sodium Phosphate Dibasic, 1.5 mM Potassium Phosphate Monobasic, 150 mM NaCl, pH 7.2; a buffer containing 20 mM sodium phosphate, pH 7.2-7.5, 350-500 mM NaCl, 3 mM MgCl2 and 1 mM phenyl methyl sulfonyl fluoride (PMSF); and the buffer optionally comprising 1 mM ADP. Any buffer may be used that is compatible with the stress protein. The preparations are then optionally purified by centrifugation through a Centricon 10 assembly (Millipore; Billerica, Mass.) to remove any unbound peptide. The non-covalent association of the proteins/peptides with the HSPs can be assayed by High Performance Liquid Chromatography (HPLC), Mass Spectrometry (MS), mixed lymphocyte target cell assay (MLTC), or enzyme-linked immunospot (ELISPOT) assay (Taguchi T, et al., J Immunol Methods 1990; 128: 65-73, incorporated herein by reference in its entirety). Once the complexes have been isolated and diluted, they can be optionally characterized further in animal models using the administration protocols and excipients described herein (see, e.g., Example 2 infra).


Complexes of stress proteins and antigenic polypeptides from separate covalent and/or non-covalent complexing reactions can be prepared to form a composition before administration to a subject. In certain embodiments, the composition is prepared within 1, 2, 3, 4, 5, 6, or 7 days before administration to a subject. In certain embodiments, the composition is prepared within 1, 2, 3, 4, 5, 6, 7, or 8 weeks before administration to a subject. In certain embodiments, the composition is prepared within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before administration to a subject. The composition can optionally be stored at about 4° C., −20° C., or −80° C. after preparation and before use.


In certain embodiments, the complexes prepared by the method disclosed herein are mixed with an adjuvant at bedside just prior to administration to a patient. In certain embodiments, the adjuvant comprises a saponin or an immunostimulatory nucleic acid. In certain embodiments, the adjuvant comprises QS-21. In certain embodiments, the dose of QS-21 is 10 μg, 25 μg, 50 μg, 75 μg, 100 μg, 125 μg, 150 μg, 175 μg, or 200 ag. In certain embodiments, the dose of QS-21 is about 100 μg. In certain embodiments, the adjuvant comprises a TLR agonist. In certain embodiments, the TLR agonist is an agonist of TLR4. In certain embodiments, the TLR agonist is an agonist of TLR7 and/or TLR8. In certain embodiments, the TLR agonist is an agonist of TLR9. In certain embodiments, the TLR agonist is an agonist of TLR5.


As an alternative to making non-covalent complexes of stress proteins and polypeptides, the polypeptides can be covalently attached to stress proteins, e.g., by chemical crosslinking or UV crosslinking. Any chemical crosslinking or UV crosslinking methods known in the art (see, e.g., Wong, 1991, Chemistry of Protein Conjugation and Cross-Linking, CRC Press, incorporated herein by reference in its entirety) can be employed. For example, glutaraldehyde crosslinking (see, e.g., Barrios et al., 1992, Eur. J. Immunol. 22: 1365-1372, incorporated herein by reference in its entirety) may be used. In an exemplary protocol, 1-2 mg of HSP-peptide complex is cross-linked in the presence of 0.002% glutaraldehyde for 2 hours. Glutaraldehyde is removed by dialysis against phosphate buffered saline (PBS) overnight (Lussow et al., 1991, Eur. J. Immunol. 21: 2297-2302, incorporated herein by reference in its entirety).


6.3.3 Vaccines

In another aspect, the instant disclosure provides a vaccine comprising the antigenic polypeptide compositions disclosed herein. The vaccine may be prepared by any method that results in a stable, sterile, preferably injectable formulation.


In certain embodiments, the vaccine comprises one or more compositions disclosed herein and one or more adjuvants. A variety of adjuvants may be employed, including, for example, systemic adjuvants and mucosal adjuvants. A systemic adjuvant is an adjuvant that can be delivered parenterally. Systemic adjuvants include adjuvants that create a depot effect, adjuvants that stimulate the immune system, and adjuvants that do both.


An adjuvant that creates a depot effect is an adjuvant that causes the antigen to be slowly released in the body, thus prolonging the exposure of immune cells to the antigen. This class of adjuvants includes alum (e.g., aluminum hydroxide, aluminum phosphate); or emulsion-based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in-water-in oil emulsion, oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.; and PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC, Pharmaceuticals Corporation, San Diego, Calif.).


Other adjuvants stimulate the immune system, for instance, cause an immune cell to produce and secrete cytokines or IgG. This class of adjuvants includes immunostimulatory nucleic acids, such as CpG oligonucleotides; saponins purified from the bark of the Q. saponaria tree, such as QS-21; poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); RNA mimetics such as polyinosinic:polycytidylic acid (poly I:C) or poly I:C stabilized with poly-lysine (poly-ICLC [Hiltonol®; Oncovir, Inc.]; derivatives of lipopolysaccharides (LPS) such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).


Other systemic adjuvants are adjuvants that create a depot effect and stimulate the immune system. These compounds have both of the above-identified functions of systemic adjuvants. This class of adjuvants includes but is not limited to ISCOMs (Immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia); AS01 which is a liposome based formulation containing MPL and QS-21 (GlaxoSmithKline, Belgium); AS02 (GlaxoSmithKline, which is an oil-in-water emulsion containing MPL and QS-21: GlaxoSmithKline, Rixensart, Belgium); AS04 (GlaxoSmithKline, which contains alum and MPL; GSK, Belgium); AS15 which is a liposome based formulation containing CpG oligonucleotides, MPL and QS-21 (GlaxoSmithKline, Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex Adjuvant Formulation (SAF, an oil-in-water emulsion containing Tween 80 and a nonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.).


The mucosal adjuvants useful according to the invention are adjuvants that are capable of inducing a mucosal immune response in a subject when administered to a mucosal surface in conjunction with complexes disclosed herein. Mucosal adjuvants include CpG nucleic acids (e.g. PCT published patent application WO 99/61056, incorporated herein by reference in its entirety), bacterial toxins: e.g., Cholera toxin (CT), CT derivatives including but not limited to CT B subunit (CTB); CTD53 (Val to Asp); CTK97 (Val to Lys); CTK104 (Tyr to Lys); CTD53/K63 (Val to Asp, Ser to Lys); CTH54 (Arg to His); CTN107 (His to Asn); CTE114 (Ser to Glu); CTE112K (Glu to Lys); CTS61F (Ser to Phe); CTS 106 (Pro to Lys); and CTK63 (Ser to Lys), Zonula occludens toxin (zot), Escherichia coli heat-labile enterotoxin, Labile Toxin (LT), LT derivatives including but not limited to LT B subunit (LTB); LT7K (Arg to Lys); LT61F (Ser to Phe); LT112K (Glu to Lys); LT118E (Gly to Glu); LT146E (Arg to Glu); LT192G (Arg to Gly); LTK63 (Ser to Lys); and LTR72 (Ala to Arg), Pertussis toxin, PT. including PT-9K/129G; Toxin derivatives (see below); Lipid A derivatives (e.g., monophosphoryl lipid A, MPL); Muramyl Dipeptide (MDP) derivatives; bacterial outer membrane proteins (e.g., outer surface protein A (OspA) lipoprotein of Borrelia burgdorferi, outer membrane protein of Neisseria meningitidis); oil-in-water emulsions (e.g., MF59; aluminum salts (Isaka et al., 1998, 1999); and Saponins (e.g., QS-21, e.g., QS-21 Stimulon®, Antigenics LLC, Lexington, Mass.), ISCOMs, MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.); the Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720; AirLiquide, Paris, France); PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC Pharmaceuticals Corporation, San Diego, Calif.); Syntext Adjuvant Formulation (SAF; Syntex Chemicals, Inc., Boulder, Colo.); poly[di(carboxylatophenoxy)]phosphazene (PCPP polymer; Virus Research Institute, USA) and Leishmania elongation factor (Corixa Corporation, Seattle, Wash.).


In certain embodiments, the adjuvant added to the compositions disclosed herein comprises a saponin and/or an immunostimulatory nucleic acid. In certain embodiments, the adjuvant added to the composition comprises or further comprises QS-21.


In certain embodiments, the adjuvant added to the compositions disclosed herein comprises a Toll-like receptor (TLR) agonist. In certain embodiments, the TLR agonist is an agonist of TLR4. In certain embodiments, the TLR agonist is an agonist of TLR7 and/or TLR8. In certain embodiments, the TLR agonist is an agonist of TLR9. In certain embodiments, the TLR agonist is an agonist of TLR5.


The compositions disclosed herein described herein may be combined with an adjuvant in several ways. For example, different polypeptides may be mixed together first to form a mixture and then complexed with stress protein(s) and/or adjuvant(s) to form a composition. As another example, different polypeptides may be complexed individually with a stress protein and/or adjuvant(s), and the resulting batches of complexes may then be mixed to form a composition.


The adjuvant can be administered prior to, during, or following administration of the compositions comprising complexes of stress protein and polypeptides. Administration of the adjuvant and the compositions can be at the same or different administration sites.


6.3.4 Unit Dosage Forms

In another aspect, the instant disclosure provides a unit dosage form of a composition (e.g., pharmaceutical composition or vaccine) disclosed herein.


The amounts and concentrations of the antigenic polypeptides, stress proteins, and/or adjuvants at which the efficacy of a vaccine disclosed herein is effective may vary depending on the chemical nature and the potency of the polypeptides, stress proteins, and/or adjuvants. Typically, the starting amounts and concentrations in the vaccine are the ones conventionally used for eliciting the desired immune response, using the conventional routes of administration, e.g., intramuscular injection. The amounts and concentrations of the peptides, conjugates, stress proteins, and/or adjuvants can then be adjusted, e.g., by dilution using a diluent, so that an effective immune response is achieved as assessed using standard methods known in the art (e.g., determined by the antibody or T-cell response to the vaccine relative to a control formulation).


In certain embodiments, the total amount of the polypeptides and stress protein in the composition is about 10 μg to 600 μg (e.g., about 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, or 500 g, optionally about 120 μg, 240 μg, or 480 μg). In certain embodiments, the total amount of the polypeptides and stress protein in the composition is about 300 μg. In certain embodiments, the amount of the stress protein in the composition is about 250 μg to 290 μg.


In certain embodiments, the amount of the stress protein in the composition is about 10 μg to 600 μg (e.g., about 50 μg, 100 ag, 200 ag, 300 ag, 400 ag, or 500 ag, optionally about 120 μg, 240 μg, or 480 μg). In certain embodiments, the amount of the stress protein in the composition is about 300 μg. The amount of the polypeptide is calculated based on a designated molar ratio and the molecular weight of the polypeptides.


In certain embodiments, the total molar amount of the polypeptides in the unit dosage form of the composition is about 0.1 to 10 nmol (e.g., about 0.1 nmol, 0.5 nmol, 1 nmol, 2 nmol, 3 nmol, 4 nmol, 5 nmol, 6 nmol, 7 nmol, 8 nmol, 9 nmol, or 10 nmol). In certain embodiments, the total molar amount of the polypeptides in the unit dosage form of the composition is about 4 nmol. In certain embodiments, the total molar amount of the polypeptides in the unit dosage form of the composition is about 5 nmol.


The molar ratio of total polypeptides to total stress proteins can be any ratio from about 0.01:1 to about 100:1, including but not limited to about 0.01:1, 0.02:1, 0.05:1. 0.1:1. 0.2:1, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 49:1, up to 100:1. In certain embodiments, the composition comprises a plurality of complexes each comprising a polypeptide and a stress protein, wherein the molar ratio of the polypeptide to the stress protein in each complex is at least about 1:1 (e.g., about 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 49:1, up to 100:1). In certain embodiments, the molar ratio of total polypeptide(s) to total stress protein(s) is about 0.5:1 to 5:1.


In certain embodiments, the molar ratio of total polypeptide(s) to total stress protein(s) is about 1:1 to 2:1. In certain embodiments, the molar ratio of total polypeptide(s) to total stress protein(s) is about 1:1, 1.25:1, or 1.5:1. Such ratios, particularly the ratios close to 1:1, are advantageous in that the composition does not comprise a great excess of free peptide(s) that is not bound to a stress protein. Since many antigenic peptides comprising MHC-binding peptides tend to comprise hydrophobic regions, an excess amount of free peptide(s) may tend to aggregate during preparation and storage of the composition. Substantial complexation with a stress protein at a molar ratio of total polypeptide(s) to total stress protein(s) close to 1:1 (e.g., 1:1, 1.25:1, 1.5:1, or 2:1) is enabled by a high binding affinity of the polypeptide to the stress protein. Accordingly, in certain embodiments, the polypeptide binds to an HSP (e.g., Hsc70, Hsp70, Hsp90, Hsp110, Grp170, Gp96, or Calreticulin) with a Kd lower than 10−3 M, 10−4 M, 10−5 M, 10−6 M, 10−7 M, 10−8 M, or 10−9 M. In certain embodiments, the polypeptide binds to Hsc70 (e.g., human Hsc70) with a Kd of 10−3 M, 10−4 M, 10−5 M, 10−6 M, 10−7 M, 10−8 M, 10−9 M, or lower.


Methods of calculating the amounts of components in the unit dosage form are provided. For example, in certain embodiments, the polypeptides have an average molecular weight of about 3 kD, and the molecular weight of Hsc70 is about 71 kD. Assuming in one embodiment that the total amount of the polypeptides and stress protein in the composition is 300 μg, and the molar ratio of the polypeptides to hsc70 is 1.5:1. The molar amount of Hsc70 can be calculated as 300 μg divided by 71 kD+1.5×3 kD, resulting in about 4.0 nmol, and the mass amount of Hsc70 can be calculated by multiplying the molar amount with 71 kD, resulting in about 280 kD. The total molar amount of the polypeptides can be calculated as 1.5×4.0 nmol, resulting in 6.0 nmol. If 10 different polypeptides are employed, the molar amount of each polypeptide is 0.60 nmol. Assuming in another embodiment that a 300 μg dose of Hsc70 is intended to be included in a unit dosage form, and the molar ratio of polypeptides to Hsc70 is 1.5:1. The total molar amount of the polypeptides can be calculated as 300 μg divided by 71 kD then times 1.5, resulting in 6.3 nmol. If 10 different polypeptides are employed, the molar amount of each polypeptide is 0.63 nmol. In cases where one or more of the variables are different from those in the examples, the quantities of the stress proteins and of the polypeptides are scaled accordingly.


It is further appreciated that the unit dosage form can optionally comprise one or more adjuvants as disclosed supra. In certain embodiments, the adjuvant comprises a saponin and/or an immunostimulatory nucleic acid. In certain embodiments, the adjuvant comprises or further comprises QS-21. In certain embodiments, the amount of QS-21 in the unit dosage form of composition is 10 μg, 25 μg, 50 μg, 75 μg, 100 μg, 125 μg, 150 μg, 175 μg, or 200 μg. In certain embodiments, the amount of QS-21 in the unit dosage form of composition is 100 μg. In certain embodiments, the adjuvant comprises a Toll-like receptor (TLR) agonist. In certain embodiments, the TLR agonist is an agonist of TLR4. In certain embodiments, the TLR agonist is an agonist of TLR7 and/or TLR8. In certain embodiments, the TLR agonist is an agonist of TLR9. In certain embodiments, the TLR agonist is an agonist of TLR5.


6.4 Methods of Use

The compositions (e.g., pharmaceutical compositions and vaccines, and unit dosage forms thereof) disclosed herein are particularly useful for inducing a cellular immune response. Stress proteins can deliver antigenic polypeptides through the cross-presentation pathway in antigen presenting cells (APCs) (e.g., macrophages and dendritic cells (DCs) via membrane receptors (mainly CD91) or by binding to Toll-like receptors, thereby leading to activation of CD8+ and CD4+ T cells. Internalization of a stress protein/antigenic polypeptide complex results in functional maturation of the APCs with chemokine and cytokine production leading to activation of natural killer cells (NK), monocytes and Th1 and Th-2-mediated immune responses.


Accordingly, in one aspect, the instant disclosure provides a method of inducing a cellular immune response to an antigenic peptide in a subject, the method comprising administering to the subject an effective amount of a composition as disclosed herein. In another aspect, the instant disclosure provides a method of treating a disease (e.g., cancer) in a subject, the method comprising administering to the subject an effective amount of a composition as disclosed herein. The compositions disclosed herein can also be used in preparing a medicament or vaccine for the treatment of a subject.


In various embodiments, such subjects can be an animal, e.g., a mammal, a non-human primate, and a human. The term “animal” includes companion animals, such as cats and dogs; zoo animals; wild animals, including deer, foxes and raccoons; farm animals, livestock and fowl, including horses, cattle, sheep, pigs, turkeys, ducks, and chickens, and laboratory animals, such as rodents, rabbits, and guinea pigs. In certain embodiments, the subject has cancer.


6.4.1 Treatment of Cancer

The compositions disclosed herein can be used alone or in combination with other therapies for the treatment of cancer. One or more of the MHC-binding peptides disclosed herein can be present in the subject's cancer cells. In certain embodiments, one or more of the MHC-binding peptides are common to or frequently found in the type and/or stage of the cancer. In certain embodiments, one or more MHC-binding peptides are found in greater than 5% of cancers. In certain embodiments, one or more of the MHC-binding peptides are specific to the cancer of the subject.


Cancers that can be treated using the compositions disclosed herein include, without limitation, a solid tumor, a hematological cancer (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and a metastatic lesion. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignancies, e.g., sarcomas and carcinomas, e.g., adenocarcinomas of the various organ systems, such as those affecting the lung, breast, ovarian, lymphoid, gastrointestinal (e.g., colon), anal, genitals and genitourinary tract (e.g., renal, urothelial, bladder cells, prostate), pharynx, CNS (e.g., brain, neural or glial cells), head and neck, skin (e.g., melanoma), and pancreas, as well as adenocarcinomas which include malignancies such as colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), cancer of the small intestine and cancer of the esophagus. The cancer may be at an early, intermediate, late stage or metastatic cancer. In certain embodiments, the cancer is associated with elevated PD-1 activity (e.g., elevated PD-1 expression).


In one embodiment, the cancer is chosen from a lung cancer (e.g., lung adenocarcinoma or a non-small cell lung cancer (NSCLC) (e.g., a NSCLC with squamous and/or non-squamous histology, or a NSCLC adenocarcinoma)), a melanoma (e.g., an advanced melanoma), a renal cancer (e.g., a renal cell carcinoma), a liver cancer (e.g., hepatocellular carcinoma or intrahepatic cholangiocellular carcinoma), a myeloma (e.g., a multiple myeloma), a prostate cancer, a breast cancer (e.g., a breast cancer that does not express one, two or all of estrogen receptor, progesterone receptor, or Her2/neu, e.g., a triple negative breast cancer), an ovarian cancer, a colorectal cancer, a pancreatic cancer, a head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC), anal cancer, gastro-esophageal cancer (e.g., esophageal squamous cell carcinoma), mesothelioma, nasopharyngeal cancer, thyroid cancer, cervical cancer, epithelial cancer, peritoneal cancer, or a lymphoproliferative disease (e.g., a post-transplant lymphoproliferative disease). In one embodiment, the cancer is NSCLC. In one embodiment, the cancer is a renal cell carcinoma. In one embodiment, the cancer is an ovarian cancer, optionally wherein the ovarian cancer is associated with human papillomavirus (HPV) infection. In a specific embodiment, the ovarian cancer is a platinum-refractory ovarian cancer.


In one embodiment, the cancer is a hematological cancer, for example, a leukemia, a lymphoma, or a myeloma. In one embodiment, the cancer is a leukemia, for example, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), acute myeloblastic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In one embodiment, the cancer is a lymphoma, for example, B cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell like (ABC) diffuse large B cell lymphoma, germinal center B cell (GCB) diffuse large B cell lymphoma, mantle cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, recurrent follicular non-Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma. In one embodiment the cancer is a myeloma, for example, multiple myeloma.


In another embodiment, the cancer is chosen from a carcinoma (e.g., advanced or metastatic carcinoma), melanoma or a lung carcinoma, e.g., a non-small cell lung carcinoma.


In one embodiment, the cancer is a lung cancer, e.g., a lung adenocarcinoma, non-small cell lung cancer or small cell lung cancer.


In one embodiment, the cancer is a melanoma, e.g., an advanced melanoma. In one embodiment, the cancer is an advanced or unresectable melanoma that does not respond to other therapies. In other embodiments, the cancer is a melanoma with a BRAF mutation (e.g., a BRAF V600 mutation). In yet other embodiments, the compositions disclosed herein is administered after treatment with an anti-CTLA-4 antibody (e.g., ipilimumab) with or without a BRAF inhibitor (e.g., vemurafenib or dabrafenib).


In another embodiment, the cancer is a hepatocarcinoma, e.g., an advanced hepatocarcinoma, with or without a viral infection, e.g., a chronic viral hepatitis.


In another embodiment, the cancer is a prostate cancer, e.g., an advanced prostate cancer.


In yet another embodiment, the cancer is a myeloma, e.g., multiple myeloma.


In yet another embodiment, the cancer is a renal cancer, e.g., a renal cell carcinoma (RCC) (e.g., a metastatic RCC, clear cell renal cell carcinoma (CCRCC) or kidney papillary cell carcinoma).


In yet another embodiment, the cancer is chosen from a lung cancer, a melanoma, a renal cancer, a breast cancer, a colorectal cancer, a leukemia, or a metastatic lesion of the cancer.


In a particular embodiment, the cancer is AML. In another particular embodiment, the cancer is colorectal cancer.


The compositions disclosed herein may be administered when a cancer is detected, or prior to or during an episode of recurrence.


Administration can begin at the first sign of cancer or recurrence, followed by boosting doses until at least symptoms are substantially abated and for a period thereafter.


In some embodiments, the compositions can be administered to a subject with cancer who has undergone tumor resection surgery that results in an insufficient amount of resected tumor tissue (e.g., less than 7 g, less than 6 g, less than 5 g, less than 4 g, less than 3 g, less than 2 g, or less than 1 g of resected tumor tissue) for production of a therapeutically effective amount of an autologous cancer vaccine comprising a representative set of antigens collected from the resected tumor tissue. See, for example, cancer vaccines described in Expert Opin. Biol. Ther. 2009 February; 9(2):179-86; incorporated herein by reference.


The compositions disclosed herein can also be used for immunization against recurrence of cancers. Prophylactic administration of a composition to an individual can confer protection against a future recurrence of a cancer.


6.4.2 Combination Therapy

Combination therapy refers to the use of compositions disclosed herein, as a first modality, with a second modality to treat cancer. Accordingly, in certain embodiments, the instant disclosure provides a method of inducing a cellular immune response to an antigenic peptide in a subject as disclosed herein, or a method of treating a disease in a subject as disclosed herein, the method comprising administering to the subject an effective amount of (a) a composition as disclosed herein and (b) a second modality.


In one embodiment, the second modality is a non-HSP modality, e.g., a modality that does not comprise HSP as a component. This approach is commonly termed combination therapy, adjunctive therapy or conjunctive therapy (the terms are used interchangeably). With combination therapy, additive potency or additive therapeutic effect can be observed. Synergistic outcomes are sought where the therapeutic efficacy is greater than additive. The use of combination therapy can also provide better therapeutic profiles than the administration of either the first or the second modality alone.


The additive or synergistic effect may allow for a reduction in the dosage and/or dosing frequency of either or both modalities to mitigate adverse effects. In certain embodiments, the second modality administered alone is not clinically adequate to treat the subject (e.g., the subject is non-responsive or refractory to the single modality), such that the subject needs an additional modality. In certain embodiments, the subject has responded to the second modality, yet suffers from side effects, relapses, develops resistance, etc., such that the subject needs an additional modality. Methods disclosed herein comprising administration of the compositions disclosed herein to such subjects to improve the therapeutic effectiveness of the second modality. The effectiveness of a treatment modality can be assayed in vivo or in vitro using methods known in the art.


In one embodiment, a lesser amount of the second modality is required to produce a therapeutic benefit in a subject. In specific embodiments, a reduction of about 10%, 20%, 30%, 40% and 50% of the amount of second modality can be achieved. The amount of the second modality, including amounts in a range that does not produce any observable therapeutic benefits, can be determined by dose-response experiments conducted in animal models by methods well known in the art.


In certain embodiments, the second modality comprises a TCR, e.g., a soluble TCR or a cell expressing a TCR. In certain embodiments, the second modality comprises a cell expressing a chimeric antigen receptor (CAR). In certain embodiments, the cell expressing the TCR or CAR is a T cell. In a particular embodiment, the TCR or CAR binds to (e.g., specifically binds to) at least one MHC-binding epitope in the composition disclosed herein.


In certain embodiments, the second modality comprises a TCR mimic antibody. In certain embodiments, the TCR mimic antibody is an antibody that specifically binds to a peptide-MHC complex. Non-limiting examples of TCR mimic antibodies are disclosed in U.S. Pat. No. 9,074,000, U.S. Publication Nos. US 2009/0304679 A1 and US 2014/0134191 A1, all of which are incorporated herein by reference in their entireties. In a particular embodiment, the TCR mimic antibody binds to (e.g., specifically binds to) at least one MHC-binding epitope in the composition disclosed herein.


In certain embodiments, the second modality comprises a checkpoint targeting agent. In certain embodiments, the checkpoint targeting agent is selected from the group consisting of an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, an antagonist anti-PD-1 antibody, an antagonist anti-TIM-3 antibody, an antagonist anti-LAG-3 antibody, an antagonist anti-CEACAM1 antibody, an agonist anti-CD137 antibody, an antagonist anti-TIGIT antibody, an antagonist anti-VISTA antibody, an agonist anti-GITR antibody, and an agonist anti-OX40 antibody.


In certain embodiments, an anti-PD-1 antibody is used as the second modality in methods disclosed herein. In certain embodiments, the anti-PD-1 antibody is nivolumab, also known as BMS-936558 or MDX1106, developed by Bristol-Myers Squibb. In certain embodiments, the anti-PD-1 antibody is pembrolizumab, also known as lambrolizumab or MK-3475, developed by Merck & Co. In certain embodiments, the anti-PD-1 antibody is pidilizumab, also known as CT-011, developed by CureTech. In certain embodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514, developed by Medimmune. In certain embodiments, the anti-PD-1 antibody is PDR001 developed by Novartis Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is REGN2810 developed by Regeneron Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is PF-06801591 developed by Pfizer. In certain embodiments, the anti-PD-1 antibody is BGB-A317 developed by BeiGene. In certain embodiments, the anti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. In certain embodiments, the anti-PD-1 antibody is SHR-1210 developed by Hengrui.


Further non-limiting examples of anti-PD-1 antibodies that may be used in treatment methods disclosed herein are disclosed in the following patents and patent applications, all of which are herein incorporated by reference in their entireties for all purposes: U.S. Pat. Nos. 6,808,710; 7,332,582; 7,488,802; 8,008,449; 8,114,845; 8,168,757; 8,354,509; 8,686,119; 8,735,553; 8,747,847; 8,779,105; 8,927,697; 8,993,731; 9,102,727; 9,205,148; U.S. Publication No. US 2013/0202623 A1; U.S. Publication No. US 2013/0291136 A1; U.S. Publication No. US 2014/0044738 A1; U.S. Publication No. US 2014/0356363 A1; U.S. Publication No. US 2016/0075783 A1; and PCT Publication No. WO 2013/033091 A1; PCT Publication No. WO 2015/036394 A1; PCT Publication No. WO 2014/179664 A2; PCT Publication No. WO 2014/209804 A1; PCT Publication No. WO 2014/206107 A1; PCT Publication No. WO 2015/058573 A1; PCT Publication No. WO 2015/085847 A1; PCT Publication No. WO 2015/200119 A1; PCT Publication No. WO 2016/015685 A1; and PCT Publication No. WO 2016/020856 A1.


In certain embodiments, an anti-PD-L1 antibody is used as the second modality in methods disclosed herein. In certain embodiments, the anti-PD-L1 antibody is atezolizumab developed by Genentech. In certain embodiments, the anti-PD-L1 antibody is durvalumab developed by AstraZeneca, Celgene and Medimmune. In certain embodiments, the anti-PD-L1 antibody is avelumab, also known as MSB0010718C, developed by Merck Serono and Pfizer. In certain embodiments, the anti-PD-L1 antibody is MDX-1105 developed by Bristol-Myers Squibb. In certain embodiments, the anti-PD-L1 antibody is AMP-224 developed by Amplimmune and GSK.


Non-limiting examples of anti-PD-L1 antibodies that may be used in treatment methods disclosed herein are disclosed in the following patents and patent applications, all of which are herein incorporated by reference in their entireties for all purposes: U.S. Pat. Nos. 7,943,743; 8,168,179; 8,217,149; 8,552,154; 8,779,108; 8,981,063; 9,175,082; U.S. Publication No. US 2010/0203056 A1; U.S. Publication No. US 2003/0232323 A1; U.S. Publication No. US 2013/0323249 A1; U.S. Publication No. US 2014/0341917 A1; U.S. Publication No. US 2014/0044738 A1; U.S. Publication No. US 2015/0203580 A1; U.S. Publication No. US 2015/0225483 A1; U.S. Publication No. US 2015/0346208 A1; U.S. Publication No. US 2015/0355184 A1; and PCT Publication No. WO 2014/100079 A1; PCT Publication No. WO 2014/022758 A1; PCT Publication No. WO 2014/055897 A2; PCT Publication No. WO 2015/061668 A1; PCT Publication No. WO 2015/109124 A1; PCT Publication No. WO 2015/195163 A1; PCT Publication No. WO 2016/000619 A1; and PCT Publication No. WO 2016/030350 A1.


In certain embodiments, a compound that targets an immunomodulatory enzyme(s) such as IDO (indoleamine-(2,3)-dioxygenase) and/or TDO (tryptophan 2,3-dioxygenase) is used as the second modality in methods disclosed herein. Therefore, in one embodiment, the compound targets an immunomodulatory enzyme(s), such as an inhibitor of indoleamine-(2,3)-dioxygenase (IDO). In certain embodiments, such compound is selected from the group consisting of epacadostat (Incyte Corp; see, e.g., WO 2010/005958 which is herein incorporated by reference in its entirety), F001287 (Flexus Biosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). In one embodiment, the compound is epacadostat. In another embodiment, the compound is F001287. In another embodiment, the compound is indoximod. In another embodiment, the compound is NLG919. In a specific embodiment, an anti-TIM-3 (e.g., human TIM-3) antibody disclosed herein is administered to a subject in combination with an IDO inhibitor for treating cancer. The IDO inhibitor as described herein for use in treating cancer is present in a solid dosage form of a composition such as a tablet, a pill or a capsule, wherein the composition includes an IDO inhibitor and a pharmaceutically acceptable excipient. As such, the antibody as described herein and the IDO inhibitor as described herein can be administered separately, sequentially or concurrently as separate dosage forms. In one embodiment, the antibody is administered parenterally, and the IDO inhibitor is administered orally. In particular embodiments, the inhibitor is selected from the group consisting of epacadostat (Incyte Corporation), F001287 (Flexus Biosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). Epacadostat has been described in PCT Publication No. WO 2010/005958, which is herein incorporated by reference in its entirety for all purposes. In one embodiment, the inhibitor is epacadostat. In another embodiment, the inhibitor is F001287. In another embodiment, the inhibitor is indoximod. In another embodiment, the inhibitor is NLG919.


In certain embodiments, the second modality comprises a different vaccine (e.g., a peptide vaccine, a DNA vaccine, or an RNA vaccine) for treating cancer. In certain embodiments, the vaccine is a heat shock protein-based tumor vaccine or a heat shock protein-based pathogen vaccine (e.g., a vaccine as described in WO 2016/183486, which is incorporated herein by reference in its entirety). In a specific embodiment, the second modality comprises a stress protein-based vaccine. For example, in certain embodiments, the second modality comprises a composition as disclosed herein that is different from the first modality. In certain embodiments, the second modality comprises a composition analogous to those disclosed herein except for having a different sequence of the HSP-binding peptide. In certain embodiments, the stress protein-based vaccine is derived from a tumor preparation, such that the immunity elicited by the vaccine is specifically directed against the unique antigenic peptide repertoire expressed by the cancer of each subject.


In certain embodiments, the second modality comprises one or more adjuvants, such as the ones disclosed supra that may be included in the vaccine formulation disclosed herein. In certain embodiments, the second modality comprises a saponin, an immunostimulatory nucleic acid, and/or QS-21. In certain embodiments, the second modality comprises a Toll-like receptor (TLR) agonist. In certain embodiments, the TLR agonist is an agonist of TLR4. In certain embodiments, the TLR agonist is an agonist of TLR7 and/or TLR8. In certain embodiments, the TLR agonist is an agonist of TLR9. In certain embodiments, the TLR agonist is an agonist of TLR5.


In certain embodiments, the second modality comprises one or more of the agents selected from the group consisting of lenalidomide, dexamethasone, interleukin-2, recombinant interferon alfa-2b, and peginterferon alfa-2b.


In certain embodiments, where the composition is used for treating a subject having cancer, the second modality comprises a chemotherapeutic or a radiotherapeutic. In certain embodiments, the chemotherapeutic agent is a hypomethylating agent (e.g., azacitidine).


The composition disclosed herein can be administered separately, sequentially, or concurrently from the second modality (e.g., chemotherapeutic, radiotherapeutic, checkpoint targeting agent, IDO inhibitor, vaccine, adjuvant, soluble TCR, cell expressing a TCR, cell expressing a CAR, and/or TCR mimic antibody), by the same or different delivery routes.


6.4.3 Dosage Regimen

The dosage of the compositions disclosed herein, and the dosage of any additional treatment modality if combination therapy is to be administered, depends to a large extent on the weight and general state of health of the subject being treated, as well as the frequency of treatment and the route of administration. Amounts effective for this use will also depend on the stage and severity of the disease and the judgment of the prescribing physician, but generally range for the initial immunization (that is, for therapeutic administration) from about 1.0 μg to about 1000 μg (1 mg) (including, for example, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 μg) of any one of the compositions disclosed herein for a 70 kg patient, followed by boosting dosages of from about 1.0 μg to about 1000 μg of the composition (including, for example, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 μg) pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL activity in the patient's blood. Regimens for continuing therapy, including site, dose and frequency may be guided by the initial response and clinical judgment. Dosage ranges and regimens for adjuvants are known to those in the art, see, e.g., Vogel and Powell, 1995, A Compendium of Vaccine Adjuvants and Excipients; M. F. Powell, M. J. Newman (eds.), Plenum Press, New York, pages 141-228.


Preferred adjuvants include QS-21, e.g., QS-21 Stimulon®, and CpG oligonucleotides. Exemplary dosage ranges for QS-21 are 1 μg to 200 μg per administration. In other embodiments, dosages for QS-21 can be 10, 25, and 50 μg per administration. In certain embodiments, the adjuvant comprises a Toll-like receptor (TLR) agonist. In certain embodiments, the TLR agonist is an agonist of TLR4. In certain embodiments, the TLR agonist is an agonist of TLR7 and/or TLR8. In certain embodiments, the TLR agonist is an agonist of TLR9. In certain embodiments, the TLR agonist is an agonist of TLR5.


In certain embodiments, the administered amount of compositions depends on the route of administration and the type of HSPs in the compositions. For example, the amount of HSP in the compositions can range, for example, from 5 to 1000 μg (1 mg) per administration. In certain embodiments, the administered amount of compositions comprising Hsc70-, Hsp70- and/or Gp96-polypeptide complexes is, for example, 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000 μg. In certain embodiments, the administered amount of the composition is in the range of about 10 to 600 μg per administration and about 5 to 100 μg if the composition is administered intradermally. In certain embodiments, the administered amount of the composition is about 5 μg to 600 μg, about 5 μg to 300 μg, about 5 μg to 150 μg, or about 5 μg to 60 μg. In certain embodiments, the administered amount of the composition is less than 100 μg. In certain embodiments, the administered amount of the composition is about 5 μg, g, 50 μg, 120 μg, 240 μg, or 480 μg. In certain embodiments, the compositions comprising complexes of stress proteins and polypeptides are purified.


In one embodiment of a therapeutic regimen, a dosage substantially equivalent to that observed to be effective in smaller non-human animals (e.g., mice or guinea pigs) is effective for human administration, optionally subject to a correction factor not exceeding a fiftyfold increase, based on the relative lymph node sizes in such mammals and in humans. Specifically, interspecies dose-response equivalence for stress proteins (or HSPs) noncovalently bound to or mixed with antigenic molecules for a human dose is estimated as the product of the therapeutic dosage observed in mice and a single scaling ratio, not exceeding a fifty-fold increase. In certain embodiment, the dosages of the composition can be much smaller than the dosage estimated by extrapolation.


The doses recited above can be given once or repeatedly, such as daily, every other day, weekly, biweekly, or monthly, for a period up to a year or over a year. Doses are preferably given once every 28 days for a period of about 52 weeks or more.


In one embodiment, the compositions are administered to a subject at reasonably the same time as an additional treatment modality or modalities. This method provides that the two administrations are performed within a time frame of less than one minute to about five minutes, or up to about sixty minutes from each other, for example, at the same doctor's visit.


In another embodiment, the compositions and an additional treatment modality or modalities are administered concurrently.


In yet another embodiment the compositions and an additional treatment modality or modalities are administered in a sequence and within a time interval such that the complexes disclosed herein, and the additional treatment modality or modalities can act together to provide an increased benefit than if they were administered alone.


In another embodiment, the compositions and an additional treatment modality or modalities are administered sufficiently close in time so as to provide the desired therapeutic or prophylactic outcome. Each can be administered simultaneously or separately, in any appropriate form and by any suitable route. In one embodiment, the complexes disclosed herein, and the additional treatment modality or modalities are administered by different routes of administration. In an alternate embodiment, each is administered by the same route of administration. The compositions can be administered at the same or different sites, e.g. arm and leg. When administered simultaneously, the compositions and an additional treatment modality or modalities may or may not be administered in admixture or at the same site of administration by the same route of administration.


In various embodiments, the compositions and an additional treatment modality or modalities are administered less than 1 hour apart, at about 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart. In other embodiments, the compositions and a vaccine composition are administered 2 to 4 days apart, 4 to 6 days apart, 1 week a part, 1 to 2 weeks apart, 2 to 4 weeks apart, one month apart, 1 to 2 months apart, or 2 or more months apart. In preferred embodiments, the compositions and an additional treatment modality or modalities are administered in a time frame where both are still active. One skilled in the art would be able to determine such a time frame by determining the half-life of each administered component.


In certain embodiments, the compositions are administered to the subject weekly for at least four weeks. In certain embodiments, after the four weekly doses, at least 2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) further doses of the compositions are administered biweekly to the subject. In certain embodiments, the compositions administered as a booster three months after the final weekly or biweekly dose. The booster that is administered every three months can be administered for the life of the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more years). In certain embodiments, the total number of doses of the compositions administered to the subject is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.


In one embodiment, the compositions and an additional treatment modality or modalities are administered within the same patient visit. In certain embodiments, the compositions are administered prior to the administration of an additional treatment modality or modalities. In an alternate specific embodiment, the compositions are administered subsequent to the administration of an additional treatment modality or modalities.


In certain embodiments, the compositions and an additional treatment modality or modalities are cyclically administered to a subject. Cycling therapy involves the administration of the compositions for a period of time, followed by the administration of a modality for a period of time and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improve the efficacy of the treatment. In such embodiments, the disclosure contemplates the alternating administration of the compositions followed by the administration of a modality 4 to 6 days later, preferable 2 to 4 days, later, more preferably 1 to 2 days later, wherein such a cycle may be repeated as many times as desired. In certain embodiments, the compositions and the modality are alternately administered in a cycle of less than 3 weeks, once every two weeks, once every 10 days or once every week. In certain embodiments, the compositions are administered to a subject within a time frame of one hour to twenty-four hours after the administration of a modality. The time frame can be extended further to a few days or more if a slow- or continuous-release type of modality delivery system is used.


6.4.4 Routes of Administration

The compositions disclosed herein may be administered using any desired route of administration. Many methods may be used to introduce the compositions described above, including but not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, mucosal, intranasal, intra-tumoral, and intra-lymph node routes. Non-mucosal routes of administration include, but are not limited to, intradermal and topical administration. Mucosal routes of administration include, but are not limited to, oral, rectal and nasal administration. Advantages of intradermal administration include use of lower doses and rapid absorption, respectively. Advantages of subcutaneous or intramuscular administration include suitability for some insoluble suspensions and oily suspensions, respectively. Preparations for mucosal administrations are suitable in various formulations as described below.


Solubility and the site of the administration are factors which should be considered when choosing the route of administration of the compositions. The mode of administration can be varied between multiple routes of administration, including those listed above.


If the compositions are water-soluble, then it may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions, preferably sterile. Alternatively, if a composition has poor solubility in aqueous solvents, then it may be formulated with a non-ionic surfactant such as Tween, or polyethylene glycol. Thus, the compositions may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, or rectal administration.


For oral administration, the composition may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such a liquid preparation may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pre-gelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art.


The compositions for oral administration may be suitably formulated to be released in a controlled and/or timed manner.


For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.


The preparation may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The preparation may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.


The preparation may also be formulated in a rectal preparation such as a suppository or retention enema, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.


In addition to the formulations described above, the preparation may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the preparation may be formulated with suitable polymeric or hydrophobic materials (for example, as emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.


For administration by inhalation, the compositions are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.


6.4.5 Patient (Subject) Evaluation

Patients treated with the compositions disclosed herein may be tested for an anti-tumor immune response. In this regard, peripheral blood from patients may be obtained and assayed for markers of anti-tumor immunity. Using standard laboratory procedures, leukocytes may be obtained from the peripheral blood and assayed for frequency of different immune cell phenotypes, HLA subtype, and function of anti-tumor immune cells.


The majority of effector immune cells in the anti-tumor response is CD8+ T cells and thus is HLA class I restricted. Using immunotherapeutic strategies in other tumor types, expansion of CD8+ cells that recognize HLA class I restricted antigens is found in a majority of patients. However, other cell types are involved in the anti-tumor immune response, including, for example, CD4+ T cells, and macrophages and dendritic cells, which may act as antigen-presenting cells. Populations of T cells (CD4+, CD8+, and Treg cells), macrophages, and antigen presenting cells may be determined using flow cytometry. HLA typing may be performed using routine methods in the art, such as methods described in Boegel et al. Genome Medicine 2012, 4:102 (seq2HLA), or using a TruSight® HLA sequencing panel (Illumina, Inc.). The HLA subtype of CD8+ T cells may be determined by a complement-dependent microcytotoxicity test.


To determine if there is an increase in anti-tumor T cell response, an enzyme linked immunospot assay may be performed to quantify the IFNγ-producing peripheral blood mononuclear cells (PBMC). This technique provides an assay for antigen recognition and immune cell function. In some embodiments, subjects who respond clinically to the vaccine may have an increase in tumor-specific T cells and/or IFNγ-producing PBMCs. In some embodiments, immune cell frequency is evaluated using flow cytometry. In some embodiments, antigen recognition and immune cell function is evaluated using enzyme linked immunospot assays.


In some embodiments, a panel of assays may be performed to characterize the immune response generated to the composition alone or given in combination with standard of care (e.g., maximal surgical resection, radiotherapy, and concomitant and adjuvant chemotherapy with temozolomide for glioblastoma multiforme). In some embodiments, the panel of assays includes one or more of the following tests: whole blood cell count, absolute lymphocyte count, monocyte count, percentage of CD4+CD3+ T cells, percentage of CD8+CD3+ T cells, percentage of CD4+CD25+FoxP3+ regulatory T cells and other phenotyping of PBL surface markers, intracellular cytokine staining to detect proinflammatory cytokines at the protein level, qPCR to detect cytokines at the mRNA level and CFSE dilution to assay T cell proliferation.


In evaluating a subject, a number of other tests may be performed to determine the overall health of the subject. For example, blood samples may be collected from subjects and analyzed for hematology, coagulation times and serum biochemistry. Hematology for CBC may include red blood cell count, platelets, hematocrit, hemoglobin, white blood cell (WBC) count, plus WBC differential to be provided with absolute counts for neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Serum biochemistry may include albumin, alkaline phosphatase, aspartate amino transferase, alanine amino transferase, total bilirubin, BUN, glucose, creatinine, potassium and sodium. Protime (PT) and partial thromboplastin time (PTT) may also be tested. One or more of the following tests may also be conducted: anti-thyroid (anti-microsomal or thyroglobulin) antibody tests, assessment for anti-nuclear antibody, and rheumatoid factor. Urinalysis may be performed to evaluated protein, RBC, and WBC levels in urine. Also, a blood draw to determine histocompatibility leukocyte antigen (HLA) status may be performed.


In some embodiments, radiologic tumor evaluations are performed one or more times throughout a treatment to evaluate tumor size and status. For example, tumor evaluation scans may be performed within 30 days prior to surgery, within 48 hours after surgery (e.g., to evaluate percentage resection), 1 week (maximum 14 days) prior to the first vaccination (e.g., as a baseline evaluation), and approximately every 8 weeks thereafter for a particular duration. MRI or CT imaging may be used. Typically, the same imaging modality used for the baseline assessment is used for each tumor evaluation visit.


6.5 Antibodies and T Cell Receptors

In another aspect, the instant disclosure provides an isolated antibody that specifically binds to an MHC-binding peptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, and/or to a complex of an MHC molecule and an MHC-binding peptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171. In certain embodiments, the antibody does not specifically bind (or binds with reduced affinity) to an unphosphorylated variant of the MHC-binding peptide, and/or to a complex of an MHC molecule and an unphosphorylated variant of the MHC-binding peptide. The antibody can be of any format known in the art or disclosed herein. In certain embodiments, the antibody is a chimeric antigen receptor. Chimeric antigen receptors are well known in the art (see e.g., Subklewe M, et al, Transfus Med Hemother 2019; 46:15-24. doi: 10.1159/000496870, which is incorporated by reference herein in its entirety).


In another aspect, the instant disclosure provides an isolated polynucleotide encoding a VH region and/or VL region of the aforementioned antibody. The isolated polynucleotide can comprise DNA and/or RNA, and/or analogues or derivatives thereof. In certain embodiments, the isolated polynucleotide is an mRNA. In certain embodiments, the isolated polynucleotide is comprised within a vector.


In another aspect, the instant disclosure provides an engineered cell, comprising the aforementioned antibody, isolated polynucleotide (e.g., mRNA), or vector. In certain embodiments, the engineered cell is a human lymphocyte, e.g., a T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, a mucosal-associated invariant T (MAiT) cell, or a natural killer (NK) cell.


In another aspect, the instant disclosure provides an isolated T cell receptor (TCR) that specifically binds to a complex of an MHC molecule and an MHC-binding peptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171. In certain embodiments, the TCR does not specifically bind (or binds with reduced affinity) to a complex of the MHC molecule and an unphosphorylated variant of the MHC-binding peptide. The TCR can be of any format known in the art or disclosed herein. In certain embodiments, the TCR is a soluble TCR. In certain embodiments, the TCR further comprises a CD3 binding moiety. In certain embodiments, the TCR is a full-length TCR.


In another aspect, the instant disclosure provides an isolated polynucleotide encoding a variable region (e.g., a Vα and/or Vβ) of the aforementioned TCR. The isolated polynucleotide can comprise DNA and/or RNA, and/or analogues or derivatives thereof. In certain embodiments, the isolated polynucleotide is an mRNA. In certain embodiments, the isolated polynucleotide is comprised within a vector.


In another aspect, the instant disclosure provides an engineered cell, comprising the aforementioned TCR, isolated polynucleotide (e.g., mRNA), or vector. In certain embodiments, the engineered cell is a human lymphocyte, e.g., a T cell, a CD8+ T cell, a CD4+ T cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, a mucosal-associated invariant T (MAiT) cell, or a natural killer (NK) cell.


6.6 Kits

Kits are also provided for carrying out the prophylactic and therapeutic methods disclosed herein. The kits may optionally further comprise instructions on how to use the various components of the kits.


In certain embodiments, the kit comprises a first container containing a composition (e.g., composition comprising stress protein(s) and antigenic polypeptide(s) disclosed herein, and a second container containing one or more adjuvants. The adjuvant can be any adjuvant disclosed herein, e.g., a saponin, an immunostimulatory nucleic acid, or QS-21 (e.g., QS-21 Stimulon®). In certain embodiments, the kit further comprises a third container containing an additional treatment modality. The kit can further comprise an instruction on the indication, dosage regimen, and route of administration of the composition, adjuvant, and additional treatment modality, e.g., as disclosed in herein.


Alternatively, the kit can comprise the stress protein(s) and antigenic polypeptide(s) of a composition disclosed herein in separate containers. In certain embodiments, the kit comprises a first container containing one or more antigenic polypeptides disclosed herein, and a second container containing a purified stress protein capable of binding to the polypeptide.


The first container can contain any number of different polypeptides. For example, in certain embodiments, the first container contains no more than 100 different polypeptides, e.g., 2-50, 2-30, 2-20, 5-20, 5-15, 5-10, or 10-15 different polypeptides. In certain embodiments, each of the different polypeptides comprises the same HSP-binding peptide and a different antigenic peptide. In certain embodiments, the total amount of the polypeptide(s) in the first container is a suitable amount for a unit dosage. In certain embodiments, the total amount of the polypeptide(s) in the first container is about 0.1 to 20 nmol (e.g., 3, 4, 5, or 6 nmol).


The second container can contain any stress protein disclosed herein. In certain embodiments, the stress protein is selected from the group consisting of Hsc70, Hsp70, Hsp90, Hsp110, Grp170, Gp96, or Calreticulin, and a mutant or fusion protein thereof. In certain embodiments, the stress protein is Hsc70 (e.g., human Hsc70). In certain embodiments, the stress protein is a recombinant protein. In certain embodiments, the total amount of the stress protein(s) in the second container is about 10 μg to 600 μg (e.g., 120 μg, 240 μg, or 480 μg). In certain embodiments, the total amount of the stress protein(s) in the second container is about 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, or 500 μg. In certain embodiments, the amount of the stress protein in the composition is about 300 μg. In certain embodiments, the total molar amount of the stress protein(s) in the second container is calculated based on the total molar amount of the polypeptide(s) in the first container, such that the molar ratio of the polypeptide(s) to the stress protein(s) is about 0.5:1 to 5:1 (e.g., about 1:1, 1.25:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1). In certain embodiments, the total amount of the stress protein(s) in the second container is an amount for multiple administrations (e.g., less than or equal to 1 mg, 3 mg, 10 mg, 30 mg, or 100 mg).


In certain embodiments, the kit further comprises an instruction for preparing a composition from the polypeptide(s) in the first container and the stress protein(s) in the second container (e.g., an instruction for the complexing reaction as disclosed herein).


In certain embodiments, the kit further comprises a third container containing one or more adjuvants. The adjuvant can be any adjuvant disclosed herein, e.g., a saponin, an immunostimulatory nucleic acid, or QS-21 (e.g., QS-21 Stimulon®). In certain embodiments, the kit further comprises a fourth container containing an additional treatment modality. The kit can further comprise an instruction on the indication, dosage regimen, and route of administration of the composition prepared from the polypeptide(s) and stress protein(s), the adjuvant, and the additional treatment modality, e.g., as disclosed herein.


In certain embodiments, the composition, polypeptide(s), stress protein(s), adjuvant(s), and additional treatment modality in the containers are present in pre-determined amounts effective to treat cancers. If desired, the compositions can be presented in a pack or dispenser device which may contain one or more unit dosage forms of the compositions. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.


EXAMPLES

The examples in this Section are offered by way of illustration, and not by way of limitation.


6.7 Example 1: Phosphopeptide Isolation and Identification

This example describes the isolation and identification of tumor-associated phosphopeptide neoantigens from cancer patient tissue samples and cancer cell line samples.


The isolation of the phosphopeptides proceeded as follows. First, HLA:peptide complexes were immunopurified from samples using a pan-HLA class I antibody. Briefly, NHS-activated sepharose beads were conjugated with anti-human HLA class I antibody (W6/32, Bio X Cell®). Cells from samples were lysed in the presence of protease and phosphatase inhibitors and then incubated with the anti-human HLA class I antibody conjugated beads. After incubation, beads were loaded onto a poly-prep column and washed. The beads were resuspended in a no-salt buffer and transferred to a 30K MWCO Amicon® ultra spin filter for removal of the buffer.


HLA-bound peptides were eluted, desalted, and concentrated using stop and go extraction (STAGE) tip containing a C18 reversed phase matrix. Briefly, isolated HLA:peptide complexes were transferred from a the 30K MWCO Amicon® ultra spin filter into a low-protein binding tube using subsequent water rinses to ensure complete transfer. The beads were centrifuged, and the resulting supernatant was loaded onto equilibrated STAGE tips. The beads were again washed, and the supernatant was loaded onto STAGE tips for 1 minute each at 3500×g to ensure loading of any peptides which had become dissociated from HLA molecules.


Next, peptides were eluted from HLA molecules with 150 μL of 10% acetic acid. Beads were centrifuged at 300×g for 30 seconds and the supernatant transferred to a low-binding tube. This process was repeated to ensure complete elution of peptides from HLA molecules and the supernatant added to the low-binding tube. The supernatant was loaded onto the STAGE tips in 150 μL aliquots at 3500×g until the entire volume had passed through. The STAGE tips were washed using three rounds of 100 μL of 1% acetic acid, and peptides subsequently eluted using a stepwise gradient of increasing acetonitrile concentrations.


Phosphopeptides were enriched by immobilized metal affinity chromatography, using immobilized iron iminodiacetic acid metal affinity chromatography (Fe-IDA IMAC). Enriched phosphopeptides were chromatographically separated and analyzed on an Orbitrap Fusion™ Lumos™ mass spectrometer using complementary fragmentation methods and sequenced using Byonic™ software.


Data analysis was performed using Xcalibur™ viewing software. Raw data files were searched using Byonic™ against the Swissprot human protein database and a phosphopeptide database containing identified phosphopeptides from previously analyzed samples. Search parameters included: no enzyme specificity, ±10 ppm precursor mass tolerance, ±0.4 Da product ion mass tolerance, and a 1% false data rate (FDR). Allowed modifications included: fixed modifications of methyl esters (aspartic acid, glutamic acid, and C-termini), and variable modifications of oxidation (methionine, tryptophan, and cysteine) and phosphorylation (serine, threonine, and tyrosine). Peptide hits from search results were confirmed by accurate mass measurement and manually confirmed by inspection of resulting tandem mass spectra for correct amino acid sequence and phosphorylation site assignment.


6.8 Example 2: Phosphopeptide Synthesis

The antigenic peptides set forth in SEQ ID NOs: 119, 120, 228, 290, 339, 424, 433, 547, 654, 657, 933, 1157, 1179, 1207, 1224, 1335, 1337, 1357, 2668, 2972, 3205, 3705, 3755, 3883, 3885, and 3905 were synthesized using standard Fmoc solid-phase chemical synthesis with pre-loaded polystyrene Wang (PS-Wang) resin in a Symphony® X automatic synthesizer (Gyros Protein Technologies Inc®). A sample of the first amino acid loaded resin from the C-terminus was placed in a dry reaction vessel and was charged to each of the 24 reaction/pre-activation vessels. The synthesizer was programmed to run the complete synthesis cycle using O-(1H-6-Chloro benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/N-methylmorpholine HCTU/NMM activation chemistry. The phosphate group was incorporated using N-α-Fmoc-O-benzyl-L-phosphoserine, N-α-Fmoc-O-benzyl-L-phosphothreonine and N-α-Fmoc-O-benzyl-L-phosphotyrosine for serine, threonine and tyrosine respectively. A 5-fold excess of amino acid, 5-fold excess of activating reagent (HCTU) and 10-fold excess of N-methyl morpholine was used for the peptide coupling reaction. The coupling reaction was performed for 10 min with double coupling cycle for any incomplete coupling throughout the synthesis. These steps were repeated until the desired sequence was obtained.


At the end of the peptide synthesis, the resin was washed with dichloromethane (DCM) and dried. Upon completion of phosphopeptide assembly, the resin was transferred to a cleavage vessel for cleavage of the peptide from the resin. The cleavage reagent (TFA:DTT:Water:TIS at 88:5:5:2 (v/w/v/v)) was mixed with the resin and stirred for 4 hours at 25° C. Crude peptides were isolated from the resin by filtration and evaporated with N2 gas, followed by precipitation with chilled diethyl ether and storage at 20° C. for 12 hours.


The precipitated peptides were centrifuged and washed twice with diethyl ether, dried, dissolved in a 1:1 (v/v) mixture of acetonitrile and water, and lyophilized to produce a crude dry powder. The crude peptides were analyzed by reverse phase HPLC with a Luna® C18 analytical column (Phenomenex®, Inc) using a water (0.1% TFA)-acetonitrile (0.1% TFA) gradient. Peptides were further purified by prep-HPLC with a preparative Luna® C18 column (Phenomenex®, Inc) using a water (0.1% TFA)-acetonitrile (0.1% TFA) gradient. Purified fractions were analyzed using analytical HPLC and pure fractions were pooled for subsequent lyophilization. Peptide purity was tested using an analytical Luna® C18-column (Phenomenex®, Inc) and identity confirmed either by LC/MS (6550 Q-TOF, Agilent Technologies®) or MSQ Plus™ (Thermo Electron®, North America).


6.9 Example 3: HLA Binding

In this example, the HLA binding affinity of selected phosphopeptides identified in Example 1 was determined. HLA haplotype specificities were determined using predictive algorithms (IEDB.org) which match the experimentally derived binding motifs of individual HLA haplotypes with specified peptide sequences. Coupling this information with the known HLA haplotypes represented within each patient sample, allowed for prediction of the haplotype(s) that presented each phosphopeptide.


Phosphopeptides were synthesized according to the methods described in Example 2.


An AlphaScreen® assay was used to evaluate the binding of peptides to HLA molecules. Donor beads conjugated with streptavidin, and acceptor beads conjugated with the anti-human HLA class I antibody W6/32, were used to assess peptide binding. Biotinylated HLAs (A*02:01, B*07:02, C*07:01, or C*07:02) were mixed with a fixed excess of β2m and the mixtures added to each well of a 384-well microplate. Serial dilutions of the synthesized phosphopeptides were added to the wells, and the resultant HLA/β2/peptide mixtures were incubated overnight at 18° C. W6/32 conjugated acceptor beads were subsequently added to the wells, and the mixture was incubated for 1 hour at 21° C. Streptavidin conjugated donor beads were then added to the wells, and the mixture was incubated for a further 1 hour at 21° C.


The microplate was read using the PerkinElmer® plate reader, and data were plotted using the Michaelis-Menten equation to determine the Kd for each phosphopeptide.


Table 5 lists the Kd of each of the selected phosphopeptides to the indicated HLAs (A*02:01, B*07:02, C*07:01, or C*07:02). NT means that binding was not tested. NB means no binding was detected. LB stands for low binding and indicates that while some binding was observed, it was below the level that would allow accurate calculation of a Kd. In each case, the phosphopeptides bound as indicated below.









TABLE 5







HLA binding characteristics of selected phosphopeptides
















Kd in
Kd in
Kd in




SEQ ID
Predicted
nM
nM
nM
Kd in nM


Peptide
NO:
HLA
A*02:01
B*07:02
C*07:01
C*07:02
















KLLsYIQRL
433
HLA-
188
NB
NT
NT




A*02:01









KLFHGsLEEL
424
HLA-
203
NB
NT
NT




A*02:01









FLsRSIPSL
228
HLA-
641
NB
NT
NT




A*02:01









QLMtLENKL
654
HLA-
231
NB
NT
NT




A*02:01









APRtPPGVTF
120
HLA-
NB
51.98
NT
NT




B*07:02









SPFLSKRsL
1157
HLA-
NB
116.28
NT
NT




B*07:02









SPRsPISPEL
1179
HLA-
NB
911
NT
NT




B*07:02









YRLsPEPTPL
1357
HLA-
NB
NB
NT
NT




C*07:02









SRKsFVFEL
1207
HLA-
NB
NB
NT
NT




B*08:01









HRVsVILKL
339
HLA-
NB
NB
NT
NT




B*14:01









QPRTPsPLVL
657
HLA-
NB
184.8
LB
LB




B*07:02





“‘s’, ‘t’ and ‘y’ indicate phosphorylated serine, threonine and tyrosine, respectively.






The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications disclosed herein in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.


All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims
  • 1. An antigenic polypeptide of 8 to 100 amino acids in length, comprising an MHC-binding peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171.
  • 2. The antigenic polypeptide of claim 1, wherein: the amino acid sequence of the MHC-binding peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171; and/orthe amino acid sequence of the antigenic polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171.
  • 3. (canceled)
  • 4. The antigenic polypeptide of claim 1, further comprising an HSP-binding peptide, optionally wherein: (i) the HSP-binding peptide comprises the amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 1), wherein X1 is omitted, N, F, or Q; X2 is W, L, or F; X3 is L or I; X4 is R, L, or K; X5 is L, W, or I; X6 is T, L, F, K, R, or W; and X7 is W, G, K, or F;(ii) the HSP-binding peptide comprises the amino acid sequence of:
  • 5.-43. (canceled)
  • 44. The antigenic polypeptide of claim 1, wherein: the MHC-binding peptide is 8 to 50 amino acids in length, optionally 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length;the C-terminus of the MHC-binding peptide is linked to the N-terminus of the HSP-binding peptide;the N-terminus of the MHC-binding peptide is linked to the C-terminus of the HSP-binding peptide;the HSP-binding peptide is linked to the MHC-binding peptide via a chemical linker; and/orthe HSP-binding peptide is linked to the MHC-binding peptide via a peptide linker, optionally wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 43, or the peptide linker comprises the amino acid sequence of FR.
  • 45.-50. (canceled)
  • 51. The antigenic polypeptide of claim 44, wherein the N-terminus of the MHC-binding peptide is linked to the C-terminus of: (a) the amino acid sequence of X1X2X3X4X5X6X7FFRK (SEQ ID NO: 68), wherein X1 is omitted, N, F, or Q; X2 is W, L, or F; X3 is L or I; X4 is R, L, or K; X5 is L, W, or I; X6 is T, L, F, K, R, or W; and X7 is W, G, K, or F;(b) the amino acid sequence of X1LX2LTX3FFRK (SEQ ID NO: 69), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;(c) the amino acid sequence of NX1LX2LTX3FFRK (SEQ ID NO: 70), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;(d) the amino acid sequence of WLX1LTX2FFRK (SEQ ID NO: 71), wherein X1 is R or K; and X2 is W or G;(e) the amino acid sequence of NWLX1LTX2FFRK (SEQ ID NO: 72), wherein X1 is R or K; and X2 is W or G;(f) the amino acid sequence of NWX1X2X3X4X5FFRK (SEQ ID NO: 73), wherein X1 is L or I; X2 is L, R, or K; X3 is L or I; X4 is T, L, F, K, R, or W; and X5 is W or K; or(g) an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-97.
  • 52.-75. (canceled)
  • 76. The isolated polypeptide of claim 44, wherein: the C-terminus of the MHC-binding peptide is linked to the N-terminus of:(a) the amino acid sequence of FFRKX1X2X3X4X5X6X7 (SEQ ID NO: 44), wherein X1 is omitted, N, F, or Q; X2 is W, L, or F; X3 is L or I; X4 is R, L, or K; X5 is L, W, or I; X6 is T, L, F, K, R, or W; and X7 is W, G, K, or F;(b) the amino acid sequence of FFRKX1LX2LTX3 (SEQ ID NO: 45), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;(c) the amino acid sequence of FFRKNX1LX2LTX3 (SEQ ID NO: 46), wherein X1 is W or F; X2 is R or K; and X3 is W, F, or G;(d) the amino acid sequence of FFRKWLX1LTX2 (SEQ ID NO: 47), wherein X1 is R or K; and X2 is W or G;(e) the amino acid sequence of FFRKNWLX1LTX2 (SEQ ID NO: 48), wherein X1 is R or K; and X2 is W or G;(f) the amino acid sequence of FFRKNWX1X2X3X4X5 (SEQ ID NO: 49), wherein X1 is L or I; X2 is L, R, or K; X3 is L or I; X4 is T, L, F, K, R, or W; and X5 is W or K; or(g) an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-67.
  • 77.-94. (canceled)
  • 95. The antigenic polypeptide of claim 1, wherein: a) the amino acid sequence of the antigenic polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1372-3919, 3997-4148, and 4172-4217;b) the antigenic polypeptide is 8 to 50 amino acids in length, optionally 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length; and/orc) the antigenic polypeptide is chemically synthesized, and/or comprises a phosphopeptide, wherein a phosphorylated amino acid residue of the phosphopeptide is replaced by a non-hydrolyzable mimetic of the phosphorylated amino acid residue.
  • 96.-99. (canceled)
  • 100. A composition comprising at least one of the antigenic polypeptides of claim 1, optionally wherein: the composition further comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 different antigenic polypeptides; and/oran adjuvant, optionally wherein the adjuvant comprises a saponin or an immunostimulatory nucleic acid, optionally QS-21, and/or a TLR agonist, optionally a TLR4 agonist, TLR5 agonist, TLR7 agonist, TLR8 agonist, and/or TLR9 agonist.
  • 101. A composition comprising a complex of the antigenic polypeptide of claim 1, and a purified stress protein, optionally wherein the stress protein is selected from the group consisting of Hsc70, Hsp70, Hsp90, Hsp110, Grp170, Gp96, Calreticulin, and a mutant or fusion protein thereof; optionally wherein the stress protein comprises human Hsc70, optionally wherein the Hsc70 comprises the amino acid sequence of SEQ ID NO: 3920; the stress protein is a recombinant protein; and/oreach of the different polypeptides comprise the same HSP-binding peptide and a different MHC-binding peptide.
  • 102.-118. (canceled)
  • 119. A method of inducing a cellular immune response to an antigenic polypeptide in a subject, the method comprising administering to the subject an effective amount of the antigenic polypeptide of claim 1.
  • 120. (canceled)
  • 121. A method of treating a disease in a subject, the method comprising administering to the subject an effective amount of the antigenic polypeptide of claim 1.
  • 122.-131. (canceled)
  • 132. A kit comprising a first container containing the polypeptide of claim 1 and a second container containing a purified stress protein capable of binding to the polypeptide.
  • 133-145. (canceled)
  • 146. A method of making a vaccine, the method comprising mixing one or more of the polypeptides of claim 1 with a purified stress protein under suitable conditions such that the purified stress protein binds to at least one of the polypeptides.
  • 147.-153. (canceled)
  • 154. An isolated antibody that: (i) specifically binds to an MHC-binding peptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, optionally wherein the antibody does not specifically bind to an unphosphorylated variant of the MHC-binding peptide; and/or (ii) specifically binds to a complex of an MHC molecule and an MHC-binding peptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, optionally wherein the antibody does not specifically bind to a complex of an MHC molecule and an unphosphorylated variant of the MHC-binding peptide.
  • 155. (canceled)
  • 156. An isolated T cell receptor (TCR) that specifically binds to a complex of an MHC molecule and an MHC-binding peptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171, optionally wherein the TCR does not specifically bind to a complex of the MHC molecule and an unphosphorylated variant of the MHC-binding peptide.
  • 157.-158. (canceled)
  • 159. An isolated polynucleotide encoding: (i) a VH and/or VL of an antibody that specifically binds to an MHC-binding peptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171; or (ii) a variable region of a TCR that specifically binds to a complex of an MHC molecule and an MHC-binding peptide selected from the group consisting of SEQ ID NOs: 98-1371, 3921-3996, and 4149-4171.
  • 160.-161. (canceled)
  • 162. A vector comprising the polynucleotide of claim 159.
  • 163. (canceled)
  • 164. An engineered cell comprising the polynucleotide of claim 159.
  • 165.-166. (canceled)
  • 167. An engineered cell comprising the antibody of claim 154.
  • 168. An engineered cell comprising the TCR of claim 156.
1. RELATED APPLICATIONS

This application is a Continuation of International Patent Application No. PCT/US2020/043435, filed on Jul. 24, 2020, which claims priority to U.S. Provisional Patent Application Ser. No. 62/878,159, entitled “Antigenic Polypeptides And Methods Of Use Thereof”, filed Jul. 24, 2019, and U.S. Provisional Patent Application Ser. No. 62/925,616, entitled “Antigenic Polypeptides And Methods Of Use Thereof”, filed Oct. 24, 2019. The contents of the aforementioned applications are hereby incorporated by reference herein in their entireties.

Provisional Applications (2)
Number Date Country
62925616 Oct 2019 US
62878159 Jul 2019 US
Continuations (1)
Number Date Country
Parent PCT/US2020/043435 Jul 2020 US
Child 17582548 US