Random peptide library presented by human leukocyte antigens

文档序号：689636 发布日期：2021-04-30 浏览：21次中文

阅读说明：本技术 由人白细胞抗原呈现的随机肽文库 (Random peptide library presented by human leukocyte antigens ) 是由 M·吉 L·西贝纳于 2019-08-30 设计创作，主要内容包括：本文描述了一种抗原筛选文库,其包含多个人白细胞抗原(HLA)-抗原多肽复合物,所述HLA-抗原多肽复合物包含：(a)HLA多肽；(b)随机抗原多肽,其包含SEQ ID NO：1至209中的任何一个所示的氨基酸序列,其中选择所述随机抗原多肽以特异性结合至HLA多肽的肽结合裂口；和(c)β2微球蛋白多肽。这些文库可用于确定能够与所选T细胞受体(TCR)相互作用和刺激所选TCR的抗原性多肽。(Described herein is an antigen screening library comprising a plurality of Human Leukocyte Antigen (HLA) -antigen polypeptide complexes comprising: (a) an HLA polypeptide; (b) a random antigen polypeptide comprising SEQ ID NO: 1 to 209, wherein the random antigen polypeptide is selected to specifically bind to the peptide binding cleft of an HLA polypeptide; and (c) a β 2 microglobulin polypeptide. These libraries can be used to identify antigenic polypeptides capable of interacting with and stimulating selected T Cell Receptors (TCRs).)

1. An antigen screening library comprising a plurality of Human Leukocyte Antigen (HLA) -antigen polypeptide complexes, the HLA-antigen polypeptide complexes comprising:

a) an HLA polypeptide comprising a peptide binding cleft;

b) a random antigen polypeptide comprising SEQ ID NO: 1 to 209, wherein the random antigen polypeptide specifically binds to the peptide binding cleft of the HLA polypeptide; and

c) a beta-2 (beta 2) microglobulin polypeptide.

2. The antigen screening library of claim 1, wherein the plurality of HLA-antigen-complexes comprise HLA polypeptides selected from the group consisting of: a3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, B57, C1, C2, C3, C4, C5, C6, C7, C8, and E.

3. The antigen screening library of claim 1, wherein the plurality of HLA-antigen complexes comprises at least 5, 10, 15, 20, or 25 different HLA polypeptides selected from the group consisting of: a3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8 and E.

4. The antigen screening library of claim 1, wherein the plurality of HLA-antigen complexes comprises all HLA polypeptides of A3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E.

5. The antigen screening library of claim 1, wherein the plurality of HLA-antigen complexes comprises a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 427 to 455, of an amino acid sequence at least 87.5%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of said HLA polypeptide.

6. The antigen screening library of any one of claims 1-5, wherein the plurality of HLA-antigen polypeptide complexes comprises a peptide comprising at least about 10⁵At least about 10 of the different random antigen polypeptides⁵Individual HLA-antigen polypeptide complexes.

7. The antigen screening library of any one of claims 1 to 6, wherein the HLA polypeptide, the random antigen polypeptide, and the β 2-microglobulin polypeptide comprise a single polypeptide.

8. The antigen screening library of claim 7, wherein the single polypeptide further comprises a first flexible polypeptide linker and a second flexible polypeptide linker.

9. The antigen screening library of claim 8, wherein the random antigen polypeptide is at the N-terminus of the HLA polypeptide on the single polypeptide and the HLA polypeptide is at the N-terminus of the β 2-microglobulin polypeptide on the single polypeptide.

10. The antigen screening library of claim 9, wherein the first flexible polypeptide linker separates the HLA polypeptide from the random antigen polypeptide and the second flexible polypeptide linker separates the HLA polypeptide from the β 2-microglobulin polypeptide.

11. The antigen screening library of claim 8, wherein the random antigen polypeptide is at the C-terminus of the HLA polypeptide on the single polypeptide and the HLA polypeptide is at the N-terminus of the β 2-microglobulin polypeptide on the single polypeptide.

12. The antigen screening library of claim 11, wherein the first flexible polypeptide linker separates the HLA polypeptide from the random antigen polypeptide and the second flexible polypeptide linker separates the HLA polypeptide from the β 2-microglobulin polypeptide.

13. The antigen screening library of claim 8, wherein the random antigen polypeptide is at the N-terminus of the HLA polypeptide on the single polypeptide and the HLA polypeptide is at the C-terminus of the β 2-microglobulin polypeptide on the single polypeptide.

14. The antigen screening library of claim 13, wherein the first flexible polypeptide linker separates the random antigen polypeptide from the β 2-microglobulin polypeptide and the second flexible polypeptide linker separates the β 2-microglobulin polypeptide from the HLA polypeptide.

15. The antigen screening library of claim 8, wherein the random antigen polypeptide is at the C-terminus of the HLA polypeptide on the single polypeptide and the HLA polypeptide is at the C-terminus of the β 2-microglobulin polypeptide on the single polypeptide.

16. The antigen screening library of claim 15, wherein the first flexible polypeptide linker separates the HLA polypeptide from the β 2-microglobulin polypeptide and the second flexible polypeptide linker separates the random antigen polypeptide from the HLA polypeptide.

17. The antigen screening library of claim 8, wherein the β 2-microglobulin polypeptide is at the C-terminus of the HLA polypeptide on the single polypeptide and the HLA polypeptide is at the N-terminus of the random antigen polypeptide on the single polypeptide.

18. The antigen screening library of claim 17, wherein the first flexible polypeptide linker separates the HLA polypeptides from the random antigen polypeptides and the second flexible polypeptide linker separates the random antigen polypeptides from the β 2-microglobulin polypeptides.

19. The antigen screening library of claim 8, wherein the random antigen polypeptide is at the C-terminus of the β 2-microglobulin on the single polypeptide and the HLA polypeptide is at the C-terminus of the random antigen polypeptide on the single polypeptide.

20. The antigen screening library of claim 19, wherein the first flexible polypeptide linker separates the β 2-microglobulin polypeptide from the random antigen polypeptide and the second flexible polypeptide linker separates the random antigen polypeptide from the HLA polypeptide.

21. The antigen screening library of any one of claims 1-20, wherein each HLA-antigen complex of the plurality of HLA-antigen complexes does not comprise an epitope tag.

22. The antigen screening library of any one of claims 1-20, wherein at least one HLA-antigen complex of the plurality of HLA-antigen complexes comprises an epitope tag.

23. The antigen screening library of any one of claims 1-20, wherein at least one HLA-antigen complex of the plurality of HLA-antigen complexes does not comprise an epitope tag and at least one HLA-antigen complex of the plurality of HLA-antigen complexes comprises an epitope tag.

24. The antigen screening library of claim 22 or 23, wherein the epitope tag comprises a FLAG tag, a c-Myc tag, a HIS tag, a Hemagglutinin (HA) tag, a VSVg tag, or a V5 tag.

25. The antigen screening library of any one of claims 1-24, wherein the HLA-antigen complexes each comprise a membrane tethering domain.

26. The antigen screening library of claim 25, wherein the membrane tethering domain comprises Aga 2.

27. The antigen screening library of any one of claims 1 to 26, wherein the antigen screening library is expressed on a plurality of cells.

28. The antigen screening library of claim 27, wherein the plurality of cells is a plurality of yeast cells.

29. The antigen screening library of claim 28, wherein the plurality of yeast cells are a plurality of yeast cells of the EBY100 strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae).

30. The antigen screening library of any one of claims 27-29, wherein each cell of the plurality of cells expresses a particular HLA-antigen complex.

31. An antigen screening library comprising a plurality of Human Leukocyte Antigen (HLA) -antigen polypeptide complexes, the HLA-antigen polypeptide complexes comprising:

a) an HLA polypeptide comprising a peptide binding cleft; and

b) a random antigen polypeptide comprising SEQ ID NO: 1 to 209, wherein the random antigenic polypeptide specifically binds to the peptide binding cleft of an HLA polypeptide.

32. The antigen screening library of claim 31, wherein the plurality of HLA-antigen complexes comprise HLA polypeptides selected from the group consisting of: a3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, B57, C1, C2, C3, C4, C5, C6, C7, C8, and E.

33. The antigen screening library of claim 31, wherein the plurality of HLA-antigen complexes comprises at least 5, 10, 15, 20, or 25 different HLA polypeptides selected from the group consisting of: a3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8 and E.

34. The antigen screening library of claim 31, wherein the plurality of HLA-antigen complexes comprises all HLA polypeptides of A3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E.

35. The antigen screening library of claim 31, wherein the plurality of HLA-antigen complexes comprises a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 427 to 455, of an amino acid sequence at least 87.5%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of said HLA polypeptide.

36. The antigen screening library of any one of claims 31-35, wherein the plurality of HLA-antigen polypeptide complexes comprises a polypeptide complex comprising at least about 10⁵At least about 10 of the different random antigen polypeptides⁵Individual HLA-antigen polypeptide complexes.

37. The antigen screening library of any one of claims 31-36, wherein the HLA polypeptides and the random antigen polypeptides comprise a single polypeptide.

38. The antigen screening library of claim 37, wherein the single polypeptide further comprises a first flexible polypeptide linker that separates the HLA polypeptide from the random antigen polypeptide.

39. The antigen screening library of claim 38, wherein the random antigen polypeptide is at the N-terminus of the HLA polypeptide on the single polypeptide.

40. The antigen screening library of claim 38, wherein the random antigen polypeptide is C-terminal to the HLA polypeptide on the single polypeptide.

41. The antigen screening library of any one of claims 31-40, wherein each HLA-antigen complex of the plurality of HLA-antigen complexes does not comprise an epitope tag.

42. The antigen screening library of any one of claims 31-40, wherein at least one HLA-antigen complex of the plurality of HLA-antigen complexes comprises an epitope tag.

43. The antigen screening library of any one of claims 31-40, wherein at least one HLA-antigen complex of the plurality of HLA-antigen complexes does not comprise an epitope tag and at least one HLA-antigen complex of the plurality of HLA-antigen complexes comprises an epitope tag.

44. The antigen screening library of claim 42 or 43, wherein the epitope tag comprises a FLAG tag, a c-Myc tag, a HIS tag, a Hemagglutinin (HA) tag, a VSVG tag, or a V5 tag.

45. The antigen screening library of any one of claims 31-44, wherein the HLA-antigen complexes each comprise a membrane tethering domain.

46. The antigen screening library of claim 45, wherein the membrane tethering domain comprises Aga 2.

47. The antigen screening library of any one of claims 31-46, wherein the antigen screening library is expressed on a plurality of cells.

48. The antigen screening library of claim 47, wherein the plurality of cells is a plurality of yeast cells.

49. The antigen screening library of claim 48, wherein the plurality of yeast cells is a plurality of yeast cells of the EBY100 strain of Saccharomyces cerevisiae.

50. The antigen screening library of any one of claims 47-49, wherein each cell of the plurality of cells expresses a particular HLA-antigen complex.

51. The antigen screening library of any one of claims 47-50, wherein each cell of the plurality of cells expresses a β 2-microglobulin polypeptide.

52. An antigen screening library comprising

a) A plurality of antigenic polypeptide- β -2(β 2) microglobulin polypeptide complexes, said antigenic polypeptide- β -2(β 2) microglobulin polypeptide complexes comprising:

i) a random antigen polypeptide comprising SEQ ID NO: 1 to 209, wherein the random antigen polypeptide specifically binds to the peptide binding cleft of an HLA polypeptide; and

ii) a β -2(β 2) microglobulin polypeptide; and

b) a plurality of HLA polypeptides constitutively expressed by one or more yeast cells and comprising a peptide binding cleft.

53. The antigen screening library of claim 52, wherein the plurality of HLA polypeptides are selected from the group consisting of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, B57, C1, C2, C3, C4, C5, C6, C7, C8, and E.

54. The antigen screening library of claim 52, wherein the plurality of HLA polypeptides comprises at least 5, 10, 15, 20, or 25 different HLA polypeptides selected from the group consisting of: a3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8 and E.

55. The antigen screening library of claim 52, wherein the plurality of HLA polypeptides comprises all of the HLA polypeptides of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E.

56. The antigen screening library of claim 52, wherein the plurality of HLA polypeptides comprises a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 427 to 455, of an amino acid sequence at least 87.5%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of said HLA polypeptide.

57. The antigen screening library of any one of claims 52-56, wherein the plurality of antigenic polypeptide-beta-2 (beta 2) microglobulin polypeptide complexesComprises at least about 10⁵At least about 10 of the different random antigen polypeptides⁵A plurality of different antigenic polypeptide-beta-2 (beta 2) microglobulin polypeptide complexes.

58. The antigen screening library of any one of claims 52-57, wherein the random antigen polypeptide and the β 2-microglobulin polypeptide comprise a single polypeptide.

59. The antigen screening library of claim 58, wherein the single polypeptide further comprises a first flexible polypeptide linker.

60. The antigen screening library of claim 59, wherein the random antigen polypeptide is at the N-terminus of the β 2-microglobulin polypeptide on the single polypeptide.

61. The antigen screening library of claim 60, wherein the random antigen polypeptide is at the C-terminus of the β 2-microglobulin polypeptide on the single polypeptide.

62. The antigen screening library of any one of claims 52-61, wherein each antigen polypeptide- β -2(β 2) microglobulin polypeptide complex of the plurality of antigen polypeptide- β -2(β 2) microglobulin polypeptide complexes does not comprise an epitope tag.

63. The antigen screening library of any one of claims 52-61, wherein at least one antigen polypeptide- β -2(β 2) microglobulin polypeptide complex of the plurality of antigen polypeptide- β -2(β 2) microglobulin polypeptide complexes comprises an epitope tag.

64. The antigen screening library of any one of claims 52-61, wherein at least one HLA-antigen complex of the plurality of HLA-antigen complexes does not comprise an epitope tag and at least one HLA-antigen complex of the plurality of HLA-antigen complexes comprises an epitope tag.

65. The antigen screening library of claim 63 or 64, wherein the epitope tag comprises a FLAG tag, a c-Myc tag, a HIS tag, a Hemagglutinin (HA) tag, a VSVG tag, or a V5 tag.

66. The antigen screening library of any one of claims 52-65, wherein the antigenic polypeptide-beta-2 (beta 2) microglobulin polypeptide complexes each comprise a membrane tethering domain.

67. The antigen screening library of claim 66, wherein the membrane tethering domain comprises Aga 2.

68. The antigen screening library of any one of claims 52-67, wherein the antigen screening library is expressed on a plurality of cells.

69. The antigen screening library of claim 68, wherein the plurality of cells is a plurality of yeast cells.

70. The antigen screening library of claim 69, wherein the plurality of yeast cells is a plurality of yeast cells of the EBY100 strain of Saccharomyces cerevisiae.

71. The antigen screening library of any one of claims 68-70, wherein each cell of the plurality of cells expresses a particular antigen polypeptide- β -2(β 2) microglobulin polypeptide complex.

72. A plurality of nucleic acids encoding the antigen screening library of any one of claims 1 to 71.

73. The plurality of nucleic acids of claim 72, wherein the HLA polypeptide consists of a sequence identical to SEQ ID NO: 456 to 484, 90%, 95%, 97%, 98%, or 99% homologous to any one of the above.

74. The plurality of nucleic acids of claim 73, wherein the random antigen polypeptide consists of SEQ ID NO: 210 to 426 in any one of the list.

75. The plurality of nucleic acids of any one of claims 72-74, wherein the plurality of nucleic acids are expressed by a plurality of cells.

76. A plurality of cells expressing the antigen screening library of any one of claims 1 to 75.

77. The plurality of cells of claim 76, wherein the plurality of cells is a plurality of yeast cells.

78. The plurality of cells of claim 77, wherein the plurality of yeast cells is a plurality of cells of the EBY100 strain of Saccharomyces cerevisiae.

79. The plurality of cells of any one of claims 76-78, wherein each cell of the plurality of cells comprises a nucleic acid of a plurality of nucleic acids encoding a particular HLA-antigen complex.

80. A method of selecting an antigen comprising contacting a plurality of cells of any one of claims 76 to 79 with a T Cell Receptor (TCR) or other macromolecule having one or more antigen binding domains.

81. The method of claim 80, wherein the TCR or other macromolecule having one or more antigen binding domains is immobilized on a substrate.

82. The method of claim 80, wherein the TCR or other macromolecule having one or more antigen binding domains is expressed by a cell.

83. The method of any one of claims 76 to 79, wherein the selecting is repeated for 2, 3, 4, or 5 cycles.

84. The method of claim 83, wherein the antigen is a polypeptide antigen.

85. The method of claim 84, wherein the antigen is a non-naturally occurring polypeptide antigen.

86. The method of claim 85, wherein said antigen is a polypeptide antigen that does not naturally occur in humans.

Background

T cells are critical for adaptive immune responses and play a role in the response to infection and cancer. T cells recognize proteins derived from foreign pathogens as well as themselves (e.g., in the case of autoimmunity). Fragments of these proteins (e.g., peptides) are presented by Human Leukocyte Antigen (HLA) molecules and are recognized by T cells via T Cell Receptors (TCRs).

Class I Major Histocompatibility (MHC) HLA molecules display peptides that result primarily from processing of endogenous antigens produced by the cell (e.g., autoantigens) as well as foreign intracellular antigens (e.g., peptides derived from viral proteins) into smaller peptides. Once the peptide is bound into the HLA peptide binding cleft, MHC class I HLA molecules interact with CD8+ cytotoxic T cells and stimulate CD8+ cytotoxic T cells. MHC class I has 3 major loci, A, B and C, where each locus is divided into a number of alleles. An allele refers to the DNA sequence of a gene at a given locus and is typically represented by at least a four-digit number (e.g., a x 24: 02), with the first letter representing the locus, the first number defining the group (or type) of alleles, and the second number defining the particular protein in the group of alleles. Second and third numbers may be appended to indicate silent and non-coding variants, respectively.

Once a particular peptide-HLA complex (pHLA) is identified, T cells are activated and may (1) be cytotoxic, (2) secrete cytokines, and/or (3) recruit other immune cells. This complex interaction between foreign or self-peptides, HLA molecules and TCRs is crucial to identify how the immune system responds molecularly to recognized pathogens. One of the biggest difficulties with this complex interaction during the immune response is to understand the specificity of the TCR based on the identity of the peptide being recognized. New methods of identifying TCRs and the phlas they recognize are needed.

Disclosure of Invention

In some embodiments, an antigen screening library provided herein comprises a plurality of Human Leukocyte Antigen (HLA) -antigen polypeptide complexes comprising (a) an HLA polypeptide comprising a peptide binding cleft, (b) a random antigen polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to 209, wherein the random antigenic polypeptide specifically binds to a peptide binding cleft of an HLA polypeptide, and (c) a β -2(β 2) microglobulin polypeptide.

In some embodiments, the plurality of HLA-antigen complexes comprises HLA polypeptides selected from the group consisting of: a3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8 and E. In some embodiments, the plurality of HLA-antigen complexes comprises at least 5, 10, 15, 20, or 25 different HLA polypeptides selected from the group consisting of: a3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8 and E. In some embodiments, the plurality of HLA-antigen-complexes comprises HLA polypeptides of all of A3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E.

In some embodiments, the plurality of HLA-antigen complexes comprises a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 427 to 455, of an amino acid sequence at least 87.5%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of said HLA polypeptide.

In some embodiments, the plurality of HLA-antigen polypeptide complexes comprises complexes comprising at least about 10⁵At least about 10 of the different random antigen polypeptides⁵Individual HLA-antigen polypeptide complexes.

In some embodiments, the HLA polypeptide, the random antigen polypeptide, and the β 2-microglobulin polypeptide comprise a single polypeptide. In some embodiments, a single polypeptide further comprises a first flexible polypeptide linker and a second flexible polypeptide linker. In some embodiments, the random antigen polypeptide is at the N-terminus of the HLA polypeptide on a single polypeptide, and the HLA polypeptide is at the N-terminus of the β 2-microglobulin polypeptide on a single polypeptide. In these embodiments, the first flexible polypeptide linker separates the HLA polypeptide from the random antigen polypeptide, and the second flexible polypeptide linker separates the HLA polypeptide from the β 2-microglobulin polypeptide. In some embodiments, the random antigen polypeptide is at the C-terminus of the HLA polypeptide on a single polypeptide, and the HLA polypeptide is at the N-terminus of the β 2-microglobulin polypeptide on a single polypeptide. In these embodiments, the first flexible polypeptide linker separates the HLA polypeptide from the random antigen polypeptide, and the second flexible polypeptide linker separates the HLA polypeptide from the β 2-microglobulin polypeptide. In some embodiments, the random antigen polypeptide is at the N-terminus of the HLA polypeptide on a single polypeptide, and the HLA polypeptide is at the C-terminus of the β 2-microglobulin polypeptide on a single polypeptide. In these embodiments, the first flexible polypeptide linker separates the random antigen polypeptide from the β 2-microglobulin polypeptide, and the second flexible polypeptide linker separates the β 2-microglobulin polypeptide from the HLA polypeptide. In some embodiments, the random antigen polypeptide is at the C-terminus of the HLA polypeptide on a single polypeptide, and the HLA polypeptide is at the C-terminus of the β 2-microglobulin polypeptide on a single polypeptide. In these embodiments, the first flexible polypeptide linker separates the HLA polypeptide from the β 2-microglobulin polypeptide, and the second flexible polypeptide linker separates the random antigen polypeptide from the HLA polypeptide. In some embodiments, the β 2-microglobulin polypeptide is C-terminal to the HLA polypeptide on a single polypeptide, and the HLA polypeptide is N-terminal to the random antigen polypeptide on a single polypeptide. In these embodiments, the first flexible polypeptide linker separates the HLA polypeptide from the random antigen polypeptide, and the second flexible polypeptide linker separates the random antigen polypeptide from the β 2-microglobulin polypeptide. In some embodiments, the random antigen polypeptide is C-terminal to the β 2-microglobulin on a single polypeptide, and the HLA polypeptide is C-terminal to the random antigen polypeptide on a single polypeptide. In these embodiments, the first flexible polypeptide linker separates the β 2-microglobulin polypeptide from the random antigen polypeptide, and the second flexible polypeptide linker separates the random antigen polypeptide from the HLA polypeptide.

In some embodiments, each HLA-antigen complex of the plurality does not comprise an epitope tag. In some embodiments, at least one HLA-antigen complex of the plurality of HLA-antigen complexes comprises an epitope tag. In some embodiments, at least one HLA-antigen complex of the plurality of HLA-antigen complexes does not comprise an epitope tag, and at least one HLA-antigen complex of the plurality of HLA-antigen complexes comprises an epitope tag. In some embodiments, the epitope tag comprises a FLAG tag, a c-Myc tag, a HIS tag, a Hemagglutinin (HA) tag, a VSVg tag, or a V5 tag.

In some embodiments, the HLA-antigen complexes each comprise a membrane tethering (tethering) domain. In some embodiments, the membrane tethering domain comprises Aga 2. In some embodiments, the antigen screening library is expressed on a plurality of cells.

In some embodiments, the plurality of cells is a plurality of yeast cells. In some embodiments, the plurality of yeast cells is a plurality of yeast cells of the EBY100 strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae).

In some embodiments, each cell of the plurality of cells expresses a specific HLA-antigen complex.

In some embodiments provided herein are antigen screening libraries comprising a plurality of Human Leukocyte Antigen (HLA) -antigen polypeptide complexes comprising an HLA polypeptide comprising a peptide binding cleft, and a random antigen polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to 209, wherein the random antigenic polypeptide specifically binds to the peptide binding cleft of the HLA polypeptide.

In some embodiments, the HLA polypeptide, the random antigen polypeptide, and the β 2-microglobulin polypeptide comprise a single polypeptide. In some embodiments, the single polypeptide further comprises a first flexible polypeptide linker separating the HLA polypeptide from the random antigen polypeptide. In certain of these embodiments, the random antigen polypeptide is at the N-terminus of the HLA polypeptide on a single polypeptide. In certain of these embodiments, the random antigen polypeptide is at the C-terminus of the HLA polypeptide on a single polypeptide.

In some embodiments, the HLA-antigen complexes each comprise a membrane tethering domain. In some embodiments, the membrane tethering domain comprises Aga 2. In some embodiments, the antigen screening library is expressed on a plurality of cells.

In some embodiments, the plurality of cells is a plurality of yeast cells. In some embodiments, the plurality of yeast cells is a plurality of yeast cells of the EBY100 strain of saccharomyces cerevisiae.

In some embodiments, each cell of the plurality of cells expresses a specific HLA-antigen complex.

In some embodiments, provided herein are antigen screening libraries comprising a plurality of antigenic polypeptide- β -2(β 2) microglobulin polypeptide complexes, antigenic polypeptide- β -2(β 2) microglobulin polypeptide complexes. In these embodiments, the antigen screening library further comprises a random antigen polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to 209, wherein the random antigen polypeptide specifically binds to the peptide binding cleft of an HLA polypeptide; and a beta-2 (. beta.2) microglobulin polypeptide. In these embodiments, the antigen screening library further comprises a plurality of HLA polypeptides constitutively expressed by the one or more yeast cells and comprising the peptide binding cleft.

In some embodiments, the plurality of antigenic polypeptide-beta-2 (beta 2) microglobulin polypeptide complexes comprises a polypeptide comprising at least about 10⁵At least about 10 of the different random antigen polypeptides⁵A plurality of different antigenic polypeptide-beta-2 (beta 2) microglobulin polypeptide complexes.

In some embodiments, the random antigen polypeptide and the β 2-microglobulin polypeptide comprise a single polypeptide. In some embodiments, the single polypeptide further comprises a first flexible polypeptide linker. In certain of these embodiments, the random antigen polypeptide is at the N-terminus of the β 2-microglobulin polypeptide on a single polypeptide. In certain of these embodiments, the random antigen polypeptide is at the C-terminus of the β 2-microglobulin polypeptide on a single polypeptide.

In some embodiments, each antigenic polypeptide- β -2(β 2) microglobulin polypeptide complex in the plurality of antigenic polypeptide- β -2(β 2) microglobulin polypeptide complexes does not comprise an epitope tag. In some embodiments, at least one antigenic polypeptide- β -2(β 2) microglobulin polypeptide complex of the plurality of antigenic polypeptide- β -2(β 2) microglobulin polypeptide complexes comprises an epitope tag. In some embodiments, at least one HLA-antigen complex of the plurality of HLA-antigen complexes does not comprise an epitope tag, and at least one HLA-antigen complex of the plurality of HLA-antigen complexes comprises an epitope tag. In some embodiments, the epitope tag comprises a FLAG tag, a c-Myc tag, a HIS tag, a Hemagglutinin (HA) tag, a VSVg tag, or a V5 tag.

In some embodiments, the antigenic polypeptide- β -2(β 2) microglobulin polypeptide complexes each comprise a membrane tethering domain. In some embodiments, the membrane tethering domain comprises Aga 2. In some embodiments, the antigen screening library is expressed on a plurality of cells.

In some embodiments, each cell of the plurality of cells expresses a specific antigenic polypeptide-beta-2 (beta 2) microglobulin polypeptide complex.

In some embodiments, provided herein are a plurality of nucleic acids encoding an antigen screening library according to the techniques of the present invention.

In some embodiments, the HLA polypeptide of the HLA-antigen complex consists of a sequence identical to SEQ ID NO: 456 to 484, 90%, 95%, 97%, 98%, or 99% homologous to any one of the above. In some embodiments, the random antigenic polypeptide of the HLA-antigen complex consists of SEQ ID NO: 210 to 426 in any one of the list.

In some embodiments, the plurality of nucleic acids are expressed by a plurality of cells.

In some embodiments, provided herein is a plurality of cells expressing an antigen screening library according to the techniques of the present invention.

In some embodiments, the plurality of cells is a plurality of yeast cells. In some embodiments, the plurality of yeast cells is a plurality of cells of the EBY100 strain of saccharomyces cerevisiae. In some embodiments, each cell of the plurality of cells comprises a nucleic acid of the plurality of nucleic acids encoding a particular HLA-antigen complex.

In some embodiments, provided herein are methods of selecting an antigen comprising contacting a plurality of cells according to the techniques of the present invention with a T Cell Receptor (TCR).

In some embodiments, the TCR is immobilized on a substrate. In some embodiments, the TCR is expressed by a cell.

In some embodiments, the repetition is selected to be 2, 3, 4, or 5 cycles.

In some embodiments, the antigen is a polypeptide antigen. In some embodiments, the antigen is a non-naturally occurring polypeptide antigen. In some embodiments, the antigen is a polypeptide antigen that does not naturally occur in humans.

Brief description of the drawings

Figure 1A shows a schematic of HLA antigen polypeptide constructs coupled to yeast cells, according to some embodiments of the present technology.

Figure 1B illustrates exemplary, non-limiting embodiments of HLA antigen polypeptide constructs tethered to cells, in accordance with some embodiments of the present technology.

Figure 2 shows an exemplary, non-limiting depiction of a process for selecting specific random antigen polypeptides that interact with specific T cell receptors, in accordance with some embodiments of the present technology.

FIGS. 3A and 3B are maps of an exemplary pCT vector (FIG. 3A) and an exemplary pYAL vector (FIG. 3B).

Figure 4 shows characterization of peptide-hla (phla) expression of multiple allotypes on the surface of yeast by flow cytometry, in accordance with some embodiments of the present technology.

Detailed Description

Described herein are antigen screening libraries for selecting and/or identifying polypeptide ligands for T Cell Receptors (TCRs). In many cases, an antigen screening library can be used to find polypeptide antigens that can interact with and stimulate human T cells as TCR ligands, including endogenous TCR antigens and non-endogenous TCR antigens, which may be novel TCR antigens and/or novel epitopes. Such neoantigens and/or neoepitopes are useful, at least for example, to stimulate one or more TCRs on T cells that may have become depleted or anergic and restore an immune response against cancer, tumors, or chronic viral infections. Thus, the present disclosure includes peptide library display, e.g., random peptide antigen libraries, in the context of a given HLA, to determine the specificity and general recognition properties of TCRs limited to HLA-mediated peptide recognition.

Once expressed using the methods described herein, a random peptide antigen library can be displayed by HLA molecules expressed on the surface of a cell. Typically, the cells displaying these HLA-antigen polypeptide complexes are not normal antigen presenting cells of the host immune system, but are cells that can be readily transformed, transfected, transduced and/or electroporated with nucleic acids encoding HLA-antigen polypeptides, including but not limited to insect cells, yeast cells and bacterial cells. In some embodiments, the library of random peptide antigens is expressed by yeast cells. Will encode at least 10⁴、10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³、10¹⁴Or 10¹⁵A mixture of plasmids of different polypeptide antigens and one or more different HLA molecules is transformed into a yeast cell. After transformation with the random peptide antigen library, yeast cells expressing a library of HLA-antigen polypeptide complexes are then contacted with a TCR or other macromolecule having one or more antigen binding domains for use as a bait. The TCRs are either (1) expressed by cells or (2) recombinantly produced, and are optionally multimerized and/or immobilized on a solid structure (e.g., beads) or by a protein scaffold (e.g., streptavidin or streptavidin-conjugated dextran (referred to as a selection reagent)). Cells expressing HLA-antigen polypeptide complexes that interact with the TCR selection reagents can be selected by an appropriate means, and after 2, 3, 4,5, 6,7 or more rounds of enrichment (e.g., cycles), nucleic acids encoding HLA-antigen polypeptide complexes can be extracted from the enriched cells and sequenced to determine polypeptide antigens that have been enriched. The enriched polypeptide antigen defines the structural attributes of interaction with a given TCR.

In some embodiments, the present disclosure includes an antigen screening library comprising a plurality of HLA-antigen polypeptide complexes. In some embodiments, the HLA-antigen polypeptide complex comprises (a) an HLA polypeptide comprising a peptide binding cleft; (b) a random antigen polypeptide comprising SEQ ID NO: 1 to 194, wherein the random antigen polypeptide is selected to specifically bind to a peptide binding cleft of an HLA polypeptide; and (c) a β -2(β 2) microglobulin polypeptide. Also provided herein are derivatives of random peptide antigens and libraries thereof, compositions thereof, pharmaceutical compositions thereof, and uses thereof. Also provided herein are nucleic acid sequences encoding one or more of the random peptide antigen libraries disclosed herein and derivatives thereof, as well as methods for expressing one or more of the random peptide antigen libraries, peptides thereof and derivatives thereof in one or more cells.

As shown in the examples provided herein, a random peptide antigen library (example 1) was designed that includes nucleic acid constructs (fig. 1A) and peptide constructs tethered to cells (e.g., yeast cells) (fig. 1B). Expression of pHLA was characterized and verified using the Yeast Display (YD) system (example 2). These phlas can interact with the TCR, and determining whether an interaction has occurred can be determined using one or more of the methods described herein, e.g., using the method shown in fig. 2. Expression of pHLA was verified by flow cytometry (example 2, level 1) and could be further functionally verified by screening a random peptide antigen library with candidate allotype-matched TCRs (example 2, level 2).

The following description of the invention is intended only to illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of the disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is to be understood that such equivalent embodiments are to be included herein.

All references listed herein are incorporated by reference in their entirety. The methods and apparatus are provided herein by way of example and are not intended to limit the present disclosure.

Certain definitions

In the following description, certain specific details are shown to provide a thorough understanding of various embodiments. However, it will be understood by those skilled in the art that the provided embodiments may be practiced without these details. Throughout the specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be interpreted in an open, inclusive sense, i.e. to mean "including but not limited to". As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. Furthermore, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

The terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length, although the number of amino acid residues can be specified (e.g., a 9mer is 9 amino acid residues). The polypeptide may comprise amino acid residues, including natural and/or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, the polypeptide may comprise modifications relative to the native or native sequence, so long as the protein retains the desired activity. These modifications may be deliberate, e.g.by site-directed mutagenesis, or may be accidental, e.g.by mutation of the host producing the protein or by error due to PCR amplification.

The term "acidic residue" refers to amino acid residues in the D-or L-form having a side chain comprising an acidic group. Exemplary acidic residues include D and E.

The term "amide residue" refers to a D-or L-form of an amino acid having a side chain of an amide derivative containing an acidic group. Exemplary residues include N and Q.

The term "aromatic residue" refers to amino acid residues in either the D-or L-form having a side chain comprising an aromatic group. Exemplary aromatic residues include F, Y and W.

The term "basic residue" refers to amino acid residues in either the D-or L-form having a side chain comprising a basic group. Exemplary basic residues include H, K and R.

The term "hydrophilic residue" refers to amino acid residues in the D-or L-form having a side chain comprising a polar group. Exemplary hydrophilic residues include C, S, T, N and Q.

The term "non-functional residue" refers to a D-or L-form of an amino acid residue having a side chain lacking an acidic, basic or aromatic group. Exemplary non-functional amino acid residues include M, G, A, V, I, L and norleucine (Nle).

The term "neutral hydrophobic residue" refers to a D-or L-form of an amino acid residue having a side chain lacking basic, acidic or polar groups. Exemplary neutral hydrophobic amino acid residues include A, V, L, I, P, W, M and F.

The term "polar hydrophobic residue" refers to amino acid residues in the D-or L-form having a side chain comprising a polar group. Exemplary polar hydrophobic amino acid residues include T, G, S, Y, C, Q and N.

The term "hydrophobic residue" refers to a D-or L-form of an amino acid residue having a side chain lacking a basic or acidic group. Exemplary hydrophobic amino acid residues include A, V, L, I, P, W, M, F, T, G, S, Y, C, Q and N.

"percent (%) sequence identity" with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignments to determine percent amino acid sequence identity can be performed in a variety of ways that are known, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software, or other software suitable for nucleic acid sequences. Suitable parameters for aligning the sequences can be determined, including the algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, the percent amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, inc and the source code has been archived with the user document under u.s.copy Office, Washington d.c.,20559, where it is registered under u.s.copy Registration No. txu 510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including digital UNIX V4.0D). All sequence comparison parameters were set by the ALIGN-2 program and were unchanged.

In the case where ALIGN-2 is used for amino acid sequence comparisons, the percentage of amino acid sequence identity for a given amino acid sequence a relative to, with, or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence a relative to, with, or against a given amino acid sequence B, having or comprising a certain percentage of amino acid sequence identity) is calculated as follows: the score X/Y is multiplied by100, where X is the number of amino acid residues scored as identical matches in the program alignment of A and B by the sequence alignment program ALIGN-2, and where Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the percent amino acid sequence identity of A to B will not be equal to the percent amino acid sequence identity of B to A. Unless otherwise specifically indicated, all amino acid sequence identity percentage values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

As used herein, the terms "homologous," "homology," or "percent homology," when used herein to describe a nucleic acid sequence relative to a reference sequence, can be determined using the formula described by Karlin & Altschul 1990 (as modified in Karlin & Altschul 1993). Such a formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul 1990. Since the filing date of this application, the percent homology of sequences can be determined using the latest version of BLAST.

"T cell receptor" (TCR) refers to an antigen/MHC binding heterodimeric protein product of a vertebrate (e.g., mammal), the TCR gene complex, including the human TCR α, β, γ, and δ chains. For example, as disclosed in Rowen 1996, the entire sequence of the human β TCR locus has been sequenced; the human TCR locus has been sequenced and re-sequenced, see, e.g., Mackelprang 2006; see, Arden 1995 for a general analysis of the T cell receptor variable gene segment family; for sequence information provided and cited in the publications, each of them is expressly incorporated herein by reference.

"decoy" refers to a TCR that binds to an antigen of the technology of the present invention or "other macromolecule having one or more antigen binding domains. Other macromolecules with one or more antigen binding domains are antibodies, darpins or synthetic molecules, including aptamers. The antigen binding domain binds to a peptide, such as one or more HLA-peptide complexes of the present technology, or a nucleic acid, such as DNA and RNA.

"exogenous" with respect to a nucleic acid or polynucleotide means that the nucleic acid is part of a recombinant nucleic acid construct or is not in its natural environment. For example, the exogenous nucleic acid may be a sequence from one species that is introduced into another species, i.e., a heterologous nucleic acid. Typically, such exogenous nucleic acids are introduced into other species by recombinant nucleic acid constructs. The exogenous nucleic acid may also be a sequence that is native to the organism and that has been reintroduced into the cells of the organism. An exogenous nucleic acid comprising a native sequence can generally be distinguished from a naturally-occurring sequence by the presence of a non-native sequence (e.g., a non-native regulatory sequence that flanks the native sequence in a recombinant nucleic acid construct) that is linked to the exogenous nucleic acid. In addition, stably transformed exogenous nucleic acids are typically integrated at positions other than the position at which the native sequence is found. Exogenous elements may be added to the construct, for example, using genetic recombination. Genetic recombination is the breaking and reassociation of DNA strands to form a new molecule of DNA encoding a new set of genetic information.

As used herein, the term "about" refers to an amount approaching 10% of the stated amount.

Structural characterization of HLA-antigen polypeptide complexes

Disclosed herein are antigen screening libraries, e.g., random peptide antigen libraries, comprising a plurality of HLA-antigen polypeptide complexes. The HLA-antigen polypeptide complexes of the present disclosure comprise at least three components: (a) a random antigen polypeptide, (b) a class I major histocompatibility (MHC I) HLA molecule, and (c) a β 2-microglobulin. In some embodiments, the random antigen polypeptide of (a) is randomized, having at least one or more conserved residues that serve as anchor residues to bind a particular type of HLA molecule. Exemplary but non-limiting random antigen polypeptide antigens and HLA types associated with them are shown in table 1 and represented by SEQ ID NO: 1 to 194 and shown in table 2 and represented by SEQ ID NO: 195 to 209. In some embodiments, the random polypeptide antigen comprises a sequence identical to, but not limited to, the sequence of SEQ ID NO: 1 to 194 and SEQ ID NO: 195 to 209, or a sequence that is at least about 70%, 75%, 80%, 85%, 87%, 87.5%, 90%, 95%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to the amino acid sequence set forth in any one of seq id nos. In some embodiments, the random polypeptide antigen comprises a sequence identical to SEQ ID NO: 1 to 194 and SEQ ID NO: 195 to 209. Also contemplated in the present disclosure are nucleic acids derived from SEQ ID NO: 1 to 194 and SEQ ID NO: an N-terminal or C-terminal truncation of any one of 195 to 209 by1, 2, 3, 4,5, 6,7, 8, 9, 10, 15, 20 or 25 amino acids. In some embodiments, the HLA molecule of (b) is an HLA polypeptide and comprises a peptide binding cleft. In some embodiments, once expressed, the random antigen polypeptide of (a) binds to the HLA polypeptide of (b) at the peptide binding cleft.

Table 1: polypeptide antigen sequence classified by HLA type

Table 2: other polypeptide antigenic sequences of HLA A11

SEQ ID NO:	Sequence of
		195	X(I/L/V)XXXXX(K/R)
196	X(I/L/V)XXXXXX(K/R)
		197	X(I/L/V)XXXXXXX(K/R)
198	X(I/L/V)XXXXXXXX(K/R)
		199	X(I/L/V)XXXXXXXXX(K/R)
200	X(Y/F)XXXXX(K/R)
		201	X(Y/F)XXXXXX(K/R)
202	X(Y/F)XXXXXXX(K/R)
		203	X(Y/F)XXXXXXXX(K/R)
204	X(Y/F)XXXXXXXXX(K/R)
		205	X(N/Y)XXXXX(K/R)
206	X(N/Y)XXXXXX(K/R)
		207	X(N/Y)XXXXXXX(K/R)
208	X(N/Y)XXXXXXXX(K/R)
		209	X(N/Y)XXXXXXXXX(K/R)

In some embodiments, an antigen screening library of the present disclosure comprises (b) at least one polypeptide consisting of, but not limited to, at least the nucleotide sequences provided in table 4 SEQ ID NOs: 210 to 411. In some embodiments, an antigen screening library of the present disclosure comprises (b) a library consisting of at least the nucleotide sequences provided in at least table 5 of SEQ ID NOs: 412 to 426. The nucleic acid encoding the random antigen polypeptide of (b) is encoded by a degenerate base sequence, thereby effectively allowing any amino acid to be encoded at a given position corresponding to the degenerate base sequence. Each random antigen polypeptide has at least one conserved anchor position, which is encoded by a restricted degenerate code or a specific sequence, which allows the random antigen polypeptide to interact more efficiently with a certain HLA type. Having at least one conserved anchor position per random antigen polypeptide increases the efficiency of forming random antigen polypeptides and HLA complexes compared to the formation of HLA complexes with completely random antigen polypeptides. In some embodiments, 1,2, or 3 amino acid residues of the random antigen polypeptide are constant. In some embodiments, the random antigen polypeptide antigen comprises a sequence identical to, but not limited to, the sequence of SEQ ID NO: 210 to 411 and SEQ ID NO: a sequence that is at least about 70%, 75%, 80%, 85%, 87%, 87.5%, 90%, 95%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of the amino acid sequences set forth in any one of 412 to 426. In some embodiments, the random antigen polypeptide antigen comprises a sequence identical to SEQ ID NO: 210 to 411 and SEQ ID NO: any one of the sequences shown in any one of 412 to 426 is identical. Also contemplated in the present disclosure are nucleic acids derived from SEQ ID NO: 210 to 411 and SEQ ID NO: a random antigen polypeptide truncation of 1,2, 3, 4,5, 6,7, 8, 9, 10, 15, 20, or 25 amino acids N-terminal or C-terminal truncation of any one of 412 to 426.

In some embodiments, the amino acid residues of the random antigen polypeptide are changed by 2, 3, or 4 different amino acids. For example, referring to table 1, the second and last position of the random antigen polypeptide that binds to HLA-a2 will comprise leucine or methionine, respectively; and leucine, methionine or valine.

The amino acid sequences in tables 1 and 2 above comprise random amino acid residues ('X') and well-defined amino acids at residues collectively referred to as anchor positions. The anchor positions specified in the library design may be changed, for example, based on the amino acid substitutions shown in table 3. One of ordinary skill in the art will appreciate that possible substitutions of X residues in the amino acid sequences of tables 1 and 2 are not limited and that additional substitutions may be included without departing from the scope of the present disclosure. For example, amino acid substitutions can be used to identify important residues of a peptide sequence that contribute to HLA binding or limit amplification of a member of the libraries described herein.

Conservative modifications will produce peptides with similar functional and chemical characteristics as the peptides subjected to such modifications. In contrast, substantial modification of the functional and/or chemical properties of the peptide can be achieved by selecting substitutions in the amino acid sequence whose effect on maintaining (a) the structure of the molecular backbone in the region of the substitution (e.g., as a sheet or helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the molecule is significantly different.

For example, a "conservative amino acid substitution" may involve the replacement of a natural amino acid residue with a non-natural residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. In addition, any natural residues in the polypeptide may also be replaced with alanine, as previously described with respect to "alanine scanning mutagenesis" (see, e.g., MacLennan 1998 and Sasaki & Sutoh 1998, which discuss alanine scanning mutagenesis).

The desired amino acid substitutions (whether conservative or non-conservative) may be determined by those skilled in the art when such substitutions are required. Exemplary amino acid substitutions are shown in table 3.

Table 3: amino acid substitution

Original residues	Exemplary permutations
		Ala(A)	Val,Leu,Ile
Arg(R)	Lys,Gln,Asn
		Asn(N)	Gln
Asp(D)	Glu
		Cys(C)	Ser,Ala
Gln(Q)	Asn
		Glu(E)	Asp
Gly(G)	Pro,Ala
		His(H)	Asn,Gln,Lys,Arg
Ile(I)	Leu, Val, Met, Ala, Phe, norleucine (Nle)
		Leu(L)	Norleucine (Nle), Ile, Val, Met, Ala, Phe
Lys(K)	Arg,1, 4-diaminobutyric acid (Dab), Gln, Asn
		Met(M)	Leu,Phe,Ile
Phe(F)	Leu,Val,Ile,Ala,Tyr
		Pro(P)	Ala
Ser(S)	Thr,Ala,Cys
		Thr(T)	Ser
Trp(W)	Tyr,Phe
		Tyr(Y)	Trp,Phe,Thr,Ser
Val(V)	Ile, Met, Leu, Phe, Ala, norleucine (Nle)

In certain embodiments, conservative amino acid substitutions also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis, rather than by synthesis in biological systems.

As indicated in the "certain definitions" section above, naturally occurring residues can be divided into several classes based on common side chain properties that may be useful for modification of the sequence. For example, a non-conservative substitution may involve exchanging a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the peptide homologous to the non-human ortholog, or into non-homologous regions of the molecule. In addition, modifications with P or G may also be used for the purpose of influencing chain orientation.

In making such modifications, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobic and charge characteristics; these are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is known in the art (Kyte & Doolittle 1982). It is known that certain amino acids may be substituted for other amino acids having similar hydropathic indices or scores and still retain similar biological activity. When the change is made based on the hydropathic index, the substitution of amino acids having a hydropathic index within. + -. 2 is preferable, those within. + -. 1 are particularly preferable, and the substitution of amino acids within. + -. 0.5 is even more particularly preferable.

It is also understood in the art that substitution of like amino acids can be made effectively based on hydrophilicity. The maximum local average hydrophilicity of a protein, determined by the hydrophilicity of its adjacent amino acids, is related to its immunogenicity and antigenicity, i.e., to the biological properties of the protein.

The following hydrophilicity values have been assigned to amino acid residues: arginine (+ 3.0); lysine (+ 3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). When changes are made based on similar hydrophilicity values, substitutions of amino acids having hydrophilicity values within ± 2 are preferred, those within ± 1 are particularly preferred, and substitutions of amino acids within ± 0.5 are even more particularly preferred. Epitopes can also be identified from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitope core regions".

Those skilled in the art will be able to determine suitable variants of the polypeptides shown in the foregoing sequences using well known techniques. To identify suitable regions of the molecule that can be altered without destroying activity, one skilled in the art can target regions that are not considered important for activity. For example, when similar polypeptides having similar activities from the same species or from other species are known, one skilled in the art can compare the amino acid sequence of a peptide to similar peptides. By such comparison, residues and portions of the molecule that are conserved between similar polypeptides can be identified. It will be appreciated that changes in regions of the peptide that are not conserved relative to such similar peptides will be less likely to adversely affect the biological activity and/or structure of the peptide. One skilled in the art will also appreciate that even in regions that are relatively conserved, naturally occurring residues can be substituted with chemically similar amino acids while maintaining activity (conservative amino acid residue substitutions). Thus, even regions that may be important for biological activity or structure may be conservatively substituted for amino acids without destroying biological activity or adversely affecting peptide structure.

In addition, one skilled in the art can review structural functional studies to identify residues in similar peptides that are important for activity or structure. In view of such a comparison, the importance of the amino acid residue in the peptide corresponding to the amino acid residue in a similar peptide that is important for activity or structure can be predicted. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues of the peptide.

One skilled in the art can also analyze three-dimensional structures and amino acid sequences associated with the structures in similar polypeptides. In view of this information, one skilled in the art can predict the alignment of amino acid residues of a peptide relative to its three-dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of a protein, as such residues may be involved in important interactions with other molecules. In addition, one skilled in the art can generate test variants comprising a single amino acid substitution at each desired amino acid residue. Variants can then be screened using activity assays known to those skilled in the art. Such data can be used to gather information about the appropriate variants. For example, variants with particular amino acid residues may be avoided if such changes are found to result in disrupted, undesirable reduced or inappropriate activity. In other words, based on information gathered from such routine experiments, the skilled person can easily determine amino acids in which further substitutions, alone or in combination with other mutations, should be avoided.

Numerous scientific publications have been devoted to the prediction of secondary structure (see, e.g., Moult 1996; Chou & Fasman 1974 a; Chou & Fasman 1974 b; Chou & Fasman 1978 a; Chou & Fasman 1978 b; and Chou & Fasman 1979). Furthermore, computer programs are currently available to assist in predicting secondary structure. One method of predicting secondary structure is based on homology modeling. For example, two polypeptides or proteins having greater than 30% sequence identity or greater than 40% similarity typically have similar structural topologies. Recent developments in protein structure databases (PDB) have provided enhanced predictability of secondary structure, including the number of potential folds within a polypeptide or protein structure (Holm & Sander 1999). It has been proposed that there is a limited number of folds in a given polypeptide or protein, and that once a critical number of structures are resolved, the accuracy of the structure prediction will be greatly improved (Brenner 1997).

Other methods of predicting secondary structure include "threading" (Jones 1997; Sippl & Flockner 1996), "contour analysis" (Bowie 1991; Gribskov 1987; Gribskov 1990), and "evolutionary association" (Holm & Sander 1999; Brenner 1997).

Table 4: nucleic acid sequences encoding random polypeptide antigens

Table 5: additional nucleic acid sequences encoding random polypeptide antigens of HLA A11

One advantage of random antigen polypeptides is that a single nucleic acid with a degenerate base code can potentially express a large number of different random antigen polypeptides, which increases the chances that any one of the screening experiments will identify one or more random antigen polypeptides that interact with a certain TCR. In some embodiments, the nucleic acid encoding the random antigen polypeptide may encode at least 1x10⁴At least 1x10⁵At least 1x10⁶At least 1x10⁷At least 1x10⁸At least 1x10⁹At least 1x10¹⁰At least 1x10¹¹At least 1x10¹²At least 1x10¹³At least 1x10¹⁴Or at least 1x10¹⁵A plurality of different random polypeptide antigens.

Peptide antigens bound in the binding cleft of HLA molecules typically have a limited length range. Most polypeptides that bind to HLA class I molecules are 8, 9, 10 or 11 amino acids in length. In some embodiments, the random antigen polypeptide that binds to an HLA molecule and forms an HLA-antigen polypeptide complex of the present disclosure is 8 to 11 amino acids in length. In some embodiments, the random antigen polypeptide is 8 to 10 amino acids in length. In some embodiments, the random antigen polypeptide is 8 amino acids in length. In some embodiments, the random antigen polypeptide is 9 amino acids in length. In some embodiments, the random antigen polypeptide is 10 amino acids in length. In some embodiments, the random antigen polypeptide is 11 amino acids in length.

Another component of the HLA-antigen polypeptide complex described herein is an HLA molecule, e.g., an HLA polypeptide. For the purposes of this disclosure, HLA molecules are class I major histocompatibility molecules. In some embodiments, the plurality of HLA polypeptides of the HLA-antigen polypeptide complexes (HLA-antigen complexes) of the present disclosure may comprise any of the following loci and alleles: a3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8 and E. In some embodiments, each HLA-antigen-complex of the plurality of HLA-antigen-complexes comprises an HLA polypeptide selected from the group consisting of: a3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8 and E. In some embodiments, the plurality of HLA-antigen complexes comprises at least 5, 10, 15, 20, or 25 different HLA polypeptides selected from the group consisting of: a3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8 and E. In some embodiments, the plurality of HLA-antigen complexes comprises all HLA polypeptides of A3, a11, a23, a24, a26, a30, a31, a33, a68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E.

In some embodiments, the amino acid sequence of an HLA polypeptide of an HLA-antigen polypeptide complex can comprise any of the amino acid sequences set forth in table 6. In some embodiments, the HLA polypeptide comprises a sequence identical to SEQ ID NO: 427 to 455, 87%, 87.5%, 90%, 95%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or 100% identical to the amino acid sequence set forth in any one of seq id nos. In some embodiments, the HLA polypeptide comprises a sequence identical to SEQ ID NO: 427 to 455 to any one of the amino acid sequences shown in any one of them. In some embodiments, the portion of the HLA polypeptide comprising the peptide binding cleft is complementary to the amino acid sequence of SEQ ID NO: 251 to 279 and a non-peptide binding cleft residue is identical to any one of SEQ ID NO: 427 to 455 is at least about 70%, 75%, 80%, 85%, 87.5%, 90%, 95%, 97%, 98%, 99% or 100% identical. Also encompassed in the present disclosure is a polypeptide selected from SEQ ID NO: 427 to 455, or a1, 2, 3, 4,5, 6,7, 8, 9, 10, 15, 20, or 25 amino acid truncation of the C-terminal truncation or N-terminal truncation of any of them.

Table 6: HLA allele amino acid sequence

The HLA polypeptides of the HLA-antigen polypeptide complex can be encoded by any of the nucleic acids set forth in table 7. In some embodiments, an HLA polypeptide is encoded by a nucleic acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% homologous to at least any one of the nucleic acid sequences listed in, but not limited to, Table 7 (e.g., SEQ ID NOS: 456 to 484). In some embodiments, the HLA polypeptide consists of a sequence identical to SEQ ID NO: 456 to 484.

Table 7: HLA allele nucleic acid sequences

In some embodiments, the plurality of HLA-antigen polypeptide complexes of the random peptide antigen library comprises at least about 10⁵Individual HLA-antigen polypeptide complexes. 10⁵The components of each of the distinct HLA-antigen polypeptide complexes collectively comprise at least about 10⁵A plurality of different random antigen polypeptides. In some embodiments, the plurality of HLA-antigen polypeptide complexes of the random peptide antigen library comprises at least about 10⁷Individual HLA-antigen polypeptide complexes. 10⁷The components of each of the distinct HLA-antigen polypeptide complexes collectively comprise at least about 10⁷A plurality of different random antigen polypeptides. In some embodiments, the plurality of HLA-antigen polypeptide complexes of the random peptide antigen library comprises at least about 10⁹Individual HLA-antigen polypeptide complexes. 10⁹The components of each of the distinct HLA-antigen polypeptide complexes collectively comprise at least about 10⁹A plurality of different random antigen polypeptides. In some embodiments, the plurality of HLA-antigen polypeptide complexes of the random peptide antigen library comprises at least about 10¹¹Individual HLA-antigen polypeptide complexes. 10¹¹The components of each of the distinct HLA-antigen polypeptide complexes collectively comprise at least about 10¹¹A plurality of different random antigen polypeptides.

In some embodiments, the plurality of HLA-antigen polypeptide complexes of the random peptide antigen library further comprise a β 2-microglobulin polypeptide that interacts with and stabilizes the HLA-antigen polypeptide complexes on the surface of the cell. The amino acid sequence of the human β 2-microglobulin polypeptide is given in NCBI seq. In some embodiments, the human β 2-microglobulin polypeptide amino acid sequence of the present disclosure is a functional naturally occurring variant of a human β 2-microglobulin polypeptide, which amino acid sequence is at least about 90%, 95%, 97%, 98%, or 99% identical to a human β 2-microglobulin polypeptide disclosed in NCBI seq.

The present disclosure also includes an antigen screening library of a plurality of HLA-antigen polypeptides, wherein β 2-microglobulin is constitutively expressed by a cell. In some embodiments, the β 2-microglobulin is encoded by a first nucleic acid, the random antigen polypeptide is encoded by a second nucleic acid, and the HLA polypeptide is encoded by a third nucleic acid. In other embodiments, the β 2-microglobulin is encoded by a first nucleic acid, and the random antigen polypeptide and the HLA polypeptide are encoded by a second nucleic acid. When encoded by the first nucleic acid, the β 2-microglobulin may be transduced, transfected or transformed into a cell before or after the second or third nucleic acid.

In some embodiments of the present disclosure, the β 2-microglobulin is fused to at least one random antigen polypeptide of the antigen screening library using techniques known to one of ordinary skill in the art. In these embodiments, the HLA polypeptide may or may not be a component of an antigen screening library. In other embodiments of the present disclosure, the at least one HLA polypeptide is fused to the at least one random antigen polypeptide of the antigen screening library using techniques known to those of ordinary skill in the art. In these embodiments, the β 2-microglobulin may be expressed by cells transduced, transfected or transformed to express other components of the antigen screening library (e.g., random antigen polypeptides and HLA polypeptides). Similar to other embodiments described herein, β 2-microglobulin is constitutively expressed by a cell. In certain of these embodiments, the cell is a yeast cell. In other embodiments, the β 2-microglobulin is not expressed by cells transduced, transfected or transformed to express other components of the antigen screening library (e.g., random antigen polypeptides and HLA polypeptides). In certain of these embodiments, the cell is a mammalian cell.

In addition to the (a) random antigen polypeptides, (b) MHC I HLA molecules, and (C) β 2-microglobulin features of the HLA-antigen polypeptide complexes of the random peptide antigen library, the HLA-antigen polypeptide complexes of the present disclosure may further comprise (d) a signal sequence, (E) a polypeptide linker between any or all of (a), (b), or (C), (f) a membrane tethering domain, and optionally (g) an epitope tag, such as a FLAG tag, a C-Myc tag, a His tag, a Hemagglutinin (HA) tag, a VSVg tag, a V5 tag, an AU1 tag, an AU5 tag, a Glu-Glu tag, an OLLAS tag, a T7 tag, a S-TagHSV tag, a KT3 tag, a TK15 tag, an Fc tag, an Xpress tag, a Ty tag, a Strep tag, an NE tag, an E tag, a C-tag, and/or AviTag. In some embodiments, the HLA-antigen complex does not comprise an epitope tag. However, in some embodiments, at least one or more of each of the plurality of HLA-antigen complexes of the random peptide antigen library comprises an epitope tag that allows confirmation of expression of at least one HLA-antigen complex using antibodies specific for the epitope. In some embodiments, each of the plurality of HLA-antigen complexes of the random peptide antigen library comprises an epitope tag.

In some embodiments, the membrane-tethering domain comprises a polypeptide linker that separates the membrane-tethering domain from one or more other features of the HLA-antigen polypeptide complex ((a) - (e) and (g)). In some embodiments, the features of the HLA-antigen polypeptide complex ((a) - (g)) are expressed as a single polypeptide. In some embodiments, (b) an HLA molecule (e.g., an HLA polypeptide), (a) a random antigen polypeptide, and (c) a β 2-microglobulin polypeptide comprise a single polypeptide. In some embodiments, (b) the HLA polypeptide and (a) the random antigen polypeptide are expressed as a single polypeptide, and (c) the β 2-microglobulin is expressed separately. For example, (c) the β 2-microglobulin may be provided by a separate polypeptide encoded by the same nucleic acid expressing (a) the random antigen polypeptide and (b) the HLA polypeptide, a separate nucleic acid, or endogenously produced by the cell thereof. In some embodiments, the random antigen polypeptide is at the N-terminus of the HLA polypeptide, and the HLA polypeptide is at the N-terminus of the β 2-microglobulin polypeptide. In some embodiments, the random antigen polypeptide is at the C-terminus of the HLA polypeptide and the HLA polypeptide is at the N-terminus of the β 2-microglobulin polypeptide. In some embodiments, the random antigen polypeptide is at the N-terminus of the HLA polypeptide and the HLA polypeptide is at the C-terminus of the β 2-microglobulin polypeptide. In some embodiments, the random antigen polypeptide is at the C-terminus of the HLA polypeptide, and the HLA polypeptide is at the C-terminus of the β 2-microglobulin polypeptide.

(a) The random antigen polypeptides, (b) the class I major histocompatibility (MHC I) HLA molecules, and (c) the β 2-microglobulin may be separated by at least one flexible polypeptide linker (e.g., a first flexible polypeptide linker, a second flexible polypeptide linker, a third flexible polypeptide linker, a fourth flexible polypeptide linker, a fifth flexible polypeptide linker, or more flexible polypeptide linkers). In some embodiments, the length of the at least one flexible polypeptide linker may range from about 3 to about 100 amino acid residues, from about 5 to about 80 amino acid residues, from about 10 to about 70 amino acid residues, from about 3 to about 100 amino acid residues, from about 20 to about 60 amino acid residues. In some embodiments, the linker may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. In some embodiments, the linker may be of formula (GGGGS)_XWherein X is 1,2, 3, 4,5, 6,7, 8, 9 or 10. In some embodiments, the linker may suitably comprise a protease cleavage site, for example a thrombin cleavage site.

In some embodiments, the HLA-antigen polypeptide complexes of the random peptide antigen library comprise a signal polypeptide that directs the HLA-antigen polypeptide complexes to the cell surface via the secretory pathway. The signal peptide is cleaved in the endoplasmic reticulum and is not expressed by the HLA-antigen polypeptide complex when located on the cell surface. The signal sequence may be any suitable sequence, such as an endogenous HLA leader sequence, or a heterologous leader sequence introduced from a different secretory or transmembrane molecule, such as an immunoglobulin leader sequence.

The HLA-antigen polypeptide complex further comprises a membrane tethering domain, such as an anchoring domain from a Glycosylphosphatidylinositol (GPI) protein and/or a domain from a yeast protein having an internal repeat sequence (PIR protein). The membrane tethering domain may comprise a transmembrane domain or a domain that interacts with a cell surface protein. In some embodiments, the membrane tethering domain comprises at least one anchor domain of a GPI protein selected from yeast Aga2, Cwp1p, Cwp2p, Aga1p, Tip1p, Flo1p, Sed1p, YCR89w, and Tir1p and/or a Pir protein selected from yeast Pir1p, Pir2p, Pir3p, Pir4p, and Pir5 p. Non-limiting examples of membrane tethering domains are provided in fig. 1B.

In other embodiments, the components of the antigen screening library of the plurality of HLA-antigen polypeptide complexes are expressed as more than one polypeptide and include cleavage sequences that separate the components of the antigen screening library of the plurality of HLA-antigen polypeptide complexes from each other. For example, the random peptide antigen is separated from the HLA polypeptide and/or from the beta-2 (beta 2) microglobulin polypeptide by a cleavage sequence. As another example, HLA peptides are separated from β -2(β 2) microglobulin polypeptides by nucleotide-encoded cleavage sequences. In some embodiments, the components of the antigen screening library are separated by more than one cleavage sequence. Suitable cleavage sequences are known to those of ordinary skill in the art and include, but are not limited to, self-cleaving peptides (P2A, T2A, F2A, and E2A), proteolytic cleavage sites (3C site, thrombin site, TEV site, factor Xa site, and EKT site), and Internal Ribosome Entry Sequences (IRES).

In some embodiments, the antigen screening library and/or HLA-antigen polypeptide complexes can be expressed by one or more cells that can be readily transfected, transduced, electroporated or transformed with the nucleic acids described herein. In some embodiments, the antigen screening library and/or HLA-antigen polypeptide complexes are expressed on a plurality of cells. In some embodiments, each cell of the plurality of cells expresses a particular HLA-antigen complex of an HLA-antigen polypeptide complex and/or another component of the antigen screening library. In some embodiments, the nucleic acid or nucleic acids encode an antigen screening library and/or HLA-antigen polypeptide complexes. In some embodiments, the antigen screening library and/or HLA-antigen polypeptide complexes comprise prokaryotic cells. In some embodiments, the cell expressing the HLA-antigen polypeptide complex comprises a eukaryotic cell. In some embodiments, the eukaryotic cell comprises a yeast cell. In some embodiments, the yeast cell is a cell of saccharomyces cerevisiae. In some embodiments, the saccharomyces cerevisiae is EBY100 strain. Transformation of Saccharomyces cerevisiae with nucleic acids can be performed by standard methodsProvided that the efficiency is sufficient to produce at least 10⁷、108、10⁹Or 10¹⁰And (4) a transformant.

In addition to the plurality of HLA-antigen polypeptide complexes of the antigen screening libraries described above, the technology of the present invention also includes at least two or more antigen screening libraries having HLA-antigen polypeptide complexes different from those described above. In some embodiments, the HLA-antigen polypeptide complex has fewer components and/or at least one different component than the plurality of HLA-antigen polypeptide complexes described above. For example, in some embodiments, the HLA-antigen polypeptide complex may further comprise (a) an HLA polypeptide having a peptide binding cleft; and (b) a random antigen polypeptide comprising a sequence of SEQ ID NO: 1 to 209. In these embodiments, the HLA polypeptide and the random antigen polypeptide comprise a single polypeptide. Furthermore, in these embodiments, the single polypeptide further comprises a first flexible polypeptide linker that separates the HLA polypeptide from the random antigen polypeptide. When expressed on a single polypeptide separated by a first flexible polypeptide linker, the random antigen polypeptide is at the N-terminus of the HLA polypeptide on the single polypeptide or the random antigen polypeptide is at the C-terminus of the HLA polypeptide on the single polypeptide.

As another example, in some embodiments, the antigen screening library of the present technology comprises (a) an HLA polypeptide constitutively expressed by one or more yeast cells, the HLA polypeptide comprising a cleft that binds to a peptide, and (b) a plurality of β -2(β 2) microglobulin polypeptide-antigen polypeptide complexes. In these embodiments, the plurality of beta-2 (beta 2) microglobulin polypeptide complexes comprise a random antigen polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to 209, wherein the random antigenic polypeptide specifically binds to a peptide binding cleft of an HLA polypeptide; and (c) a β -2(β 2) microglobulin polypeptide. In these embodiments, the random antigen polypeptide and the β 2-microglobulin polypeptide comprise a single polypeptide. Furthermore, in these embodiments, the single polypeptide further comprises a first flexible polypeptide linker that separates the beta-2 (beta 2) microglobulin polypeptide from the random antigen polypeptide. When expressed on a single polypeptide separated by a first flexible polypeptide linker, the random antigen polypeptide is at the N-terminus of the beta-2 (beta 2) microglobulin polypeptide on the single polypeptide or the random antigen polypeptide is at the C-terminus of the beta-2 (beta 2) microglobulin polypeptide on the single polypeptide.

Nucleic acid encoding HLA-antigen polypeptide complex

Also disclosed herein are nucleic acids encoding HLA-antigen polypeptide complexes of the antigen screening libraries. Nucleic acids encoding HLA-antigen polypeptide complexes of the present disclosure minimally encode: (a) a random antigen polypeptide, (b) an MHC I HLA molecule, and (c) a β 2-microglobulin. In addition to the features of (a) random antigen polypeptides, (b) MHC I HLA molecules, and (c) β 2-microglobulin of HLA-antigen polypeptide complexes of a random peptide antigen library encoded by one or more nucleic acids, HLA-antigen polypeptide complexes of the present disclosure also include nucleic acids encoding: (d) a signal sequence, (E) a polypeptide linker between (a), (B) or (C), (f) a membrane-tethering domain and optionally (g) an epitope tag, such as a FLAG tag, a C-Myc tag, a HIS tag, a hemagglutinin tag, a VSVg tag, a V5 tag, an AU1 tag, an AU5 tag, a Glu-Glu tag, an OLLAS tag, a T7 tag, an S tag, an HSV tag, a KT3 tag, a TK15 tag, an Fc tag, an Xpress tag, a Ty tag, a Strep tag, an NE tag, an E tag, a C tag and/or AviTag (fig. 1A and 1B).

In some embodiments, the nucleic acid encoding the membrane tethering domain of (f) may further encode (e) one or more polypeptide linkers that separate the membrane tethering domain from other features of the HLA-antigen polypeptide complex. In some embodiments, the nucleic acid encodes one or more flexible polypeptide linkers that separate (a) the HLA polypeptide from (b) the random antigen polypeptide and (c) the β 2-microglobulin polypeptide when all three features are encoded on a single nucleic acid.

In some embodiments, the nucleic acid encoding the single polypeptide further comprises nucleotides encoding a first flexible polypeptide linker and a second flexible polypeptide linker, wherein the nucleotide sequence encoding the first flexible polypeptide linker separates the nucleotide sequence encoding the HLA polypeptide from the nucleotide sequence encoding the random antigen polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates the nucleotide sequence encoding the random antigen polypeptide from the nucleotide sequence encoding the β 2-microglobulin polypeptide. In some embodiments, once expressed, the random antigen polypeptide is at the N-terminus of the HLA polypeptide on a single polypeptide and the HLA polypeptide is at the N-terminus of the β 2-microglobulin polypeptide on a single polypeptide.

In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates a nucleotide sequence encoding an HLA polypeptide from a nucleotide sequence encoding a random antigen polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates a nucleotide sequence encoding an HLA polypeptide from a nucleotide sequence encoding a β 2-microglobulin polypeptide. In some embodiments, once expressed, the random antigen polypeptide is C-terminal to the HLA polypeptide on a single polypeptide and the HLA polypeptide is N-terminal to the β 2-microglobulin polypeptide on a single polypeptide.

In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates a nucleotide sequence encoding an HLA polypeptide from a nucleotide sequence encoding a random antigen polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates a nucleotide sequence encoding an HLA polypeptide from a nucleotide sequence encoding a β 2-microglobulin polypeptide. In some embodiments, once expressed, the random antigen polypeptide is at the N-terminus of the HLA polypeptide on a single polypeptide and the HLA polypeptide is at the C-terminus of the β 2-microglobulin polypeptide on a single polypeptide.

In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates a nucleotide sequence encoding the random antigen polypeptide from a nucleotide sequence encoding the β 2-microglobulin polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates a nucleotide sequence encoding the β 2-microglobulin polypeptide from a nucleotide sequence encoding the HLA polypeptide. In some embodiments, once expressed, the random antigen polypeptide is C-terminal to the HLA polypeptide on a single polypeptide and the HLA polypeptide is C-terminal to the β 2-microglobulin polypeptide on a single polypeptide.

In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates a nucleotide sequence encoding an HLA polypeptide from a nucleotide sequence encoding a β 2-microglobulin polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates a nucleotide sequence encoding a random antigen polypeptide from a nucleotide sequence encoding an HLA polypeptide. In some embodiments, once expressed, the β 2-microglobulin polypeptide is C-terminal to the HLA polypeptide on a single polypeptide, and the HLA polypeptide is N-terminal to the random antigen polypeptide on a single polypeptide.

In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates the nucleotide sequence encoding the HLA polypeptide from the nucleotide sequence encoding the random antigen polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates the nucleotide sequence encoding the random antigen polypeptide from the nucleotide sequence encoding the β 2-microglobulin polypeptide. In some embodiments, once expressed, the random antigen polypeptide is C-terminal to the β 2-microglobulin on a single polypeptide and the HLA polypeptide is C-terminal to the random antigen polypeptide on a single polypeptide. In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates the nucleotide sequence encoding the β 2-microglobulin polypeptide from the nucleotide sequence encoding the random antigen polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates the nucleotide sequence encoding the random antigen polypeptide from the nucleotide sequence encoding the HLA polypeptide.

In other embodiments, the components of an antigen screening library of multiple HLA-antigen polypeptide complexes are expressed as more than one polypeptide, albeit encoded by a single nucleic acid. In these embodiments, the nucleotide-encoded cleavage sequences separate the components of the antigen screening library of the plurality of HLA-antigen polypeptide complexes from each other. For example, once expressed, the random peptide antigen is separated from the HLA polypeptide and/or from the beta-2 (beta 2) microglobulin polypeptide by a cleavage sequence. As another example, upon expression, HLA peptides are separated from β -2(β 2) microglobulin polypeptides by nucleotide-encoded cleavage sequences. In these embodiments, a portion of the HLA polypeptides are expressed separately from the other components of the antigen screening library of the plurality of HLA-antigen polypeptide complexes and, when expressed separately, are naturally paired intracellularly with the other components of the HLA-antigen polypeptide complexes.

In some embodiments, the random antigenic polypeptide of the HLA-antigen complex consists of SEQ ID NO: 210 to 411. In some embodiments, the HLA polypeptide of the HLA-antigen complex consists of a sequence identical to SEQ ID NO: 210 to 411, at least 70%, 75%, 80%, 85%, 87.5%, 90%, 95%, 97%, 98%, 99%, or 100% homologous. In some embodiments, the random antigenic polypeptide of the HLA-antigen complex consists of SEQ ID NO: 412 to 426 in a nucleic acid sequence. In some embodiments, the HLA polypeptide of the HLA-antigen complex consists of a sequence identical to SEQ ID NO: 280 to 308, at least 70%, 75%, 80%, 85%, 87.5%, 90%, 95%, 97%, 98%, 99% or 100% homologous. In some embodiments, one or more of the nucleic acids, e.g., SEQ ID NOs: one or more of the nucleic acids of 210 to 411 and 412 to 426 are expressed by a plurality of cells. In some embodiments, each cell of the plurality of cells comprises a nucleic acid encoding an HLA-antigen complex. In some embodiments, the plurality of cells is a plurality of yeast cells. In some embodiments, the plurality of yeast cells is a plurality of cells of the EBY100 strain of saccharomyces cerevisiae.

Nucleic acids encoding one or more components of an HLA-antigen polypeptide complex can be delivered to a plurality of cells using a nucleic acid or vector (e.g., an exogenous nucleic acid or exogenous vector). Suitable exogenous nucleic acids and exogenous vectors include plasmids, Bacterial Artificial Chromosomes (BACs), Yeast Artificial Chromosomes (YACs), transposons, and viral vectors. These exogenous nucleic acids and exogenous vectors may further comprise components that allow for the replication of nucleic acids encoding one or more components of an HLA-antigen polypeptide complex, components that allow for antibiotic selection to allow for the selection of cells or other organisms that express nucleic acids encoding one or more components of an HLA-antigen polypeptide complex, genes that supplement yeast autotrophs to select yeast transformants that express nucleic acids encoding one or more components of an HLA-antigen polypeptide complex, promoters or enhancers for prokaryotic or eukaryotic expression of HLA-antigen polypeptide complexes, polyadenylation sites, or marker genes that allow for visualization of transformed cells. In some embodiments, a nucleic acid comprising a nucleic acid encoding an HLA-antigen polypeptide complex of the present disclosure comprises an inducible promoter.

Methods of using HLA-antigen polypeptide complexes and nucleic acids encoding such complexes minimally include contacting one or more cells (e.g., a plurality of cells) expressing HLA-antigen polypeptide complexes with a TCR and selecting one or more cells that interact with the TCR. For example, selection can be performed by using the TCR in a "panning step" to capture one or more cells expressing the HLA-antigen polypeptide complex that interact with the TCR and wash away any non-interacting cells (e.g., one or more cells not expressing the HLA-antigen polypeptide complex that do not interact with the TCR). Nucleic acids from the interacting cells can be harvested and sequenced to elucidate the amino acid sequence of the random antigen polypeptide that interacts with the TCR. These nucleic acids can be re-transfected, transformed or transduced into one or more different cells for another round of selection. The method can be repeated for any number of selection rounds, e.g., 1,2, 3, 4,5 or more rounds (e.g., in cycles), to enrich for HLA-antigen polypeptide complexes that strongly interact with the TCR.

Sequencing platforms that may be used in the present disclosure include, but are not limited to: pyrosequencing, sequencing by synthesis, single molecule sequencing, second generation sequencing, nanopore sequencing, sequencing by ligation or sequencing by hybridization. Preferred sequencing platforms are those commercially available from Illumina (RNA-Seq) and Helicos (Digital Gene Expression or "DGE"). "next generation" sequencing methods include, but are not limited to, the following commercial methods: 1)454/Roche Lifesciences, including but not limited to Margulies 2005 and U.S. Pat. No. 7,244,559; 7,335,762, respectively; 7,211,390, respectively; 7,244,567, respectively; 7,264,929, respectively; and 7,323,305; 2) helicos Biosciences Corporation (Cambridge, MA), as described in U.S. patent nos. 7,501,245; 7,491,498, respectively; and 7,276,720; and in U.S. patent publication nos. 2006/0024711; 2009/0061439, respectively; 2008/0087826, respectively; 2006/0286566, respectively; 2006/0024711, respectively; 2006/0024678, respectively; 2008/0213770, respectively; and 2008/0103058; 3) applied Biosystems (e.g., SOLiD sequencing); 4) dover Systems (e.g., Polonator G.007 sequencing); 5) illumina, as described in U.S. patent nos. 5,750,341; 6,306,597; and 5,969,119; and 6) Pacific Biosciences, as described in U.S. Pat. Nos. 7,462,452; 7,476,504, respectively; 7,405,281, respectively; 7,170,050, respectively; 7,462,468, respectively; 7,476,503; 7,315,019, respectively; 7,302,146, respectively; and 7,313,308; and in U.S. patent publication nos. 2009/0029385; 2009/0068655, respectively; 2009/0024331, respectively; and 2008/0206764.

Method

Described herein are methods of using the HLA-antigen polypeptide complexes of the present disclosure to select or enrich for antigens that bind to a TCR (e.g., a specific TCR). In some embodiments, the methods comprise contacting one or more (e.g., a plurality of) cells expressing an HLA antigen polypeptide complex with a TCR to select an antigen using one or more libraries of transgenic HLA-antigen polypeptide cells (e.g., a library of transgenic HLA-antigen polypeptide yeast cells). The methods described herein include methods for constructing one or more libraries of yeast cells that are transgenic for HLA-antigen polypeptides.

After constructing the one or more libraries of yeast cells for transgenic HLA-antigen polypeptides, the method further comprises validating the one or more libraries of yeast cells for transgenic HLA-antigen polypeptides using a limiting dilution method comprising limiting dilution of one or more cultures of expanded yeast cells each expressing at least one HLA-antigen polypeptide using a nutrient-deficient yeast culture medium. In some embodiments, the method further comprises counting yeast from the diluted yeast culture and estimating to have at least about 10⁶、10⁷、10⁸Or 10⁹A diverse HLA-antigen polypeptide yeast cell library of individual unique HLA-antigen polypeptide sequences (e.g., clones). In some embodiments, expression of an epitope tag of a yeast cell is measured to determine 10⁶、10⁷、10⁸Or 10⁹Whether any of the individual clones are displayed on the yeast cell surface. For example, expression of the epitope tag can be determined as a surrogate value for total HLA-antigen polypeptide expression in a plurality of yeast cells, and the percentage of expression can be calculated. In some embodiments, the expression percentage is phaseEstimation of the number of yeast cells expressing certain HLA-antigen polypeptides for a library of HLA-antigen polypeptide sequences.

Referring to fig. 2, a plurality of cells 201, such as yeast, can be transformed, transfected, or electroporated with a plurality of nucleic acids 202 encoding HLA-antigen polypeptide complexes of the disclosure. The plurality of cells expressing the plurality of nucleic acids encoding HLA-antigen peptide complexes are referred to as a transgenic HLA-antigen polypeptide cell library 203. The transgenic HLA-antigen polypeptide cell library 203 is expanded by cell proliferation and expression of HLA-antigen polypeptide complexes 204 of the plurality of cells is induced by methods known in the art, for example, by galactose, lactose or isopropyl β -D-1-thiogalactopyranoside (IPTG). The TCR 205 is used to positively select cells expressing the HLA-antigen polypeptide complex that interact with the TCR. In some embodiments, the TCR is immobilized on a substrate. In some embodiments, the TCR is expressed by a cell or a plurality of cells. This selection process shown in figure 2 can be repeated for any number of selection rounds, e.g., 1,2, 3, 4,5 or more times, to obtain a single or small number of HLA-antigen polypeptide complexes that interact with the TCR. In some embodiments, the HLA-antigen polypeptide complex comprises a polypeptide antigen. In some embodiments, the polypeptide antigen is a non-naturally occurring polypeptide antigen, e.g., a polypeptide antigen that is not naturally occurring in a human. After each round of selection or after the last round of selection, the nucleic acids extracted from the selected cells 205 can be subjected to deep sequencing or next generation sequencing reactions.

In some embodiments, screening greater than at least 1x10 using methods of the present disclosure (e.g., those shown in fig. 2)⁴At least 1x10⁵At least 1x10⁶At least 1x10⁷At least 1x10⁸At least 1x10⁹At least 1x10¹⁰At least 1x10¹¹At least 1x10¹²At least 1x10¹³At least 1x10¹⁴Or at least 1x10¹⁵Individual HLA-antigen polypeptide complexes. In some embodiments, the methods of the present disclosure result in identifying less than 10⁴、10³、10²10, 9, 8, 7, 6,5, 4, 3 or 2 different HLA-antigen polypeptide complexesA compound (I) is provided. In some embodiments, greater than 90%, 95%, 97%, 98%, or 99% of the HLA-antigen polypeptide complexes remaining after 1,2, 3, 4, or 5 rounds of selection comprise less than 10, 9, 8, 7, 6,5, 4, 3, or 2 different HLA-antigen polypeptide complexes. In some embodiments, greater than 90%, 95%, 97%, 98%, or 99% of the HLA-antigen polypeptide complexes remaining after 1,2, 3, 4, or 5 rounds of selection comprise less than 10, 9, 8, 7, 6,5, 4, 3, or 2 different antigen polypeptide sequences in the HLA-antigen polypeptide complexes. In some embodiments, greater than 90%, 95%, 97%, 98%, or 99% of the HLA-antigen polypeptide complexes remaining after 1,2, 3, 4, or 5 rounds of selection comprise a single HLA-antigen polypeptide complex. In some embodiments, greater than 90%, 95%, 97%, 98%, or 99% of the HLA-antigen polypeptide complexes remaining after 1,2, 3, 4, or 5 rounds of selection comprise a single antigen polypeptide sequence in a single HLA-antigen polypeptide complex.

Expression of a naive yeast library (e.g., an HLA-antigen polypeptide sequence library described herein) minimally expresses about 15% of the total antigen polypeptide sequences (Gee2018b) in the antigen polypeptide sequence library of a single 9mer length peptide presented by HLA-a1, and at least about 5% of a single length peptide (e.g., 8mer) (Gee2018 a) in the antigen polypeptide sequence library of mixed length peptides (e.g., 8mer, 9mer, 10mer, 11mer, 12mer) presented by HLA-a 2. Despite less than about 5% of single length expression of the library of antigen polypeptide sequences with peptides of 8mer length, the TCR isolated the target 8mer antigen from the library of antigen polypeptide sequences that stimulated the TCR in an in vitro co-culture assay (Gee2018 a). Libraries of these antigenic polypeptide sequences have been screened and peptides directed against TCRs of known specificity have been isolated (Gee2018 a). Although the minimum expression level necessary for a functional library has not been determined, the data show that less than 15% expression can result in a library of antigenic polypeptide sequences useful in the methods described herein.

In some embodiments, the methods of the present disclosure further comprise identifying a polypeptide antigen that interacts with a TCR. For example, a method for determining a TCR-interacting polypeptide antigen can comprise any of the following steps:

1. production and HLA-antigen polypeptide complex construct design: in some embodiments, step (1) includes, but is not limited to, generating one or more DNA constructs and/or designed to display one or more HLA polypeptides having a naturally occurring protein sequence, a synthetic protein sequence, or a combination thereof.

2. Testing of expression of HLA-antigen polypeptide complex constructs by yeast expression: in some embodiments, step (2) includes, but is not limited to, transforming one or more electrically or chemically competent yeasts with plasmids encoding a single peptide or a peptide library (including an HLA, e.g., an HLA polypeptide, of interest). This plasmid was designed for a single peptide construct or a library of peptide constructs to display yeast proteins from the N-terminus of Aga 2. In some embodiments, expression confirmation may include antibody staining of epitope tags (e.g., V5, VSVg, c-Myc, HA) or fluorescent TCR tetramer, dimer, or dextromer (dextramer) staining of yeast displaying a single peptide-HLA construct or library of peptide-HLA constructs.

3. Optional validation step for HLA display: in some embodiments, step (3) includes, but is not limited to, antibody-based staining of the epitope tags of step (2) or fluorescent TCR tetramer, dimer, or dextromer staining of yeast displaying a single peptide-HLA construct or library of peptide-HLA constructs. In some embodiments, validation may also include staining the peptide-HLA constructs with TCRs having known specificity or used to select diverse peptide libraries presented by HLA.

4. Optional steps to re-engineer HLA for display: in some embodiments, step (4) includes, but is not limited to, random mutagenesis via an error-prone polymerase, followed by electroporation into chemically and/or electrocompetent yeast. Yeast cells expressing one or more libraries of the techniques of the invention are selected using cell separation by magnetic cell sorting (MACS) or Fluorescence Activated Cell Sorting (FACS) based on the TCR of interest. In some embodiments, isolated yeast clones are sequenced or deep sequenced to identify any functional HLA mutants that suitably display the antigenic peptide of interest. If the construct or library is not properly displayed, step (4) is included in some embodiments.

5. Generation of peptide-HLA libraries: in some embodiments, step (5) includes, but is not limited to, randomly encoded peptide ligands or explicitly encoded peptide ligands. For example, randomly encoded peptide ligands or explicitly encoded peptide ligands are uniquely designed for each HLA allele based on the preference of the peptide that each HLA allele can present. In some embodiments, step (5) further comprises producing genetic material from one or more polymerase chain reactions.

6. Selection of peptide-HLA libraries with TCRs of interest: in some embodiments, step (6) includes, but is not limited to, repeating the MACS-based or FACS-based selection. For example, a TCR of interest or other macromolecule having one or more antigen binding domains acts as a bait and can be multimerized on magnetic beads, streptavidin, dextran, or other substrates suitable for multimerization. In some embodiments, the output of the one or more selection rounds comprises physical separation of the one or more yeast cells from the TCR. After isolation, the yeast is propagated and re-induced for protein expression. These repeated rounds enrich the population of binding yeasts.

7. Deep sequencing and data analysis: this process may involve extracting genetic information from a yeast library and selecting, and sequencing the products to identify the nature of the peptides from the selected library. These data can then be analyzed to identify potential targets for the TCR, and/or input into algorithms to make predictions about TCR specificity.

T Cell Receptor (TCR)

The antigens of the transgenic HLA-antigen polypeptide cell libraries and HLA-antigen polypeptide complexes described herein can be used in conjunction with a given TCR. For example, a TCR or other macromolecule having one or more antigen-binding domains is a positive selection agent or decoy, and once bound to an antigen (e.g., an HLA-antigen polypeptide complex), its cognate antigen is identified. The TCRs described herein can be native or exogenous (e.g., recombinant) and expressed by cells, such as primary T cells, immortalized T cells, or non-T cells. In some embodiments, the TCR is immobilized on a solid support such as a column, polystyrene plate, or well or bead of a multi-well plate. In some embodiments, the TCR is polymerized as a plurality of TCRs immobilized on a bead. For example, the TCR may be multimerized on, but not limited to, magnetic beads, streptavidin, or dextran.

In some embodiments, the TCR is a soluble protein comprising at least one or more binding domains of a TCR of interest (e.g., TCRa/β, TCRy/δ). The soluble protein may be single chain or heterodimeric. In some embodiments, the soluble TCR is modified by the addition of a biotin receptor peptide sequence at the C-terminus of one polypeptide. Following biotinylation on the receptor peptide, the TCR may be multimerized or added to a substrate by binding to a biotin binding partner (e.g., avidin, streptavidin, traptavidin, neutravidin, etc.). In some embodiments, the biotin binding partner can include a detectable label, such as a fluorophore, a mass label, or the like, or can be bound to a particle (e.g., a paramagnetic particle). Selection of ligands that bind to the TCR may be performed by flow cytometry, magnetic selection, etc., as known in the art.

To the extent that the foregoing material and/or any other material incorporated by reference conflicts with the present disclosure, the present disclosure controls.

The following examples provide further representative embodiments of the technology disclosed herein.

Examples

The following examples are provided to further illustrate embodiments of the present technology and should not be construed as limiting the scope of the present technology. To the extent that certain embodiments or features thereof are mentioned, they are for illustrative purposes only, and unless otherwise specified, they are not intended to limit the technology of the present invention. Those skilled in the art can develop equivalent means without implementing the inventive ability and without departing from the technical scope of the present invention. It will be appreciated that many variations in the procedures described herein may be made while remaining within the scope of the present techniques. Such variations are intended to be included within the scope of the technology disclosed herein. Accordingly, embodiments of the techniques disclosed herein are described in the following representative examples.

Example 1: design of antigen polypeptide libraries

This example describes the design of an antigen library of the present disclosure for use with polypeptide antigen HLA complexes. Using data from known HLA binding epitope ligands from websites such as www.IEDB.org/, an exemplary algorithm for designing and selecting anchor residues for each HLA allele is as follows:

step 1: a list of polypeptides binding to a given allele is downloaded, which may contain hundreds or thousands of peptides.

Step 2: based on the downloaded known peptides, a frequency matrix of residues at each position of the peptide is constructed.

And step 3: the composition of the "anchor" used for library design was selected by using a cut-off of the first 4 residues at each position.

Example 2: electroporation of pHLA libraries

This example describes electroporation of yeast cells with nucleic acids encoding an exemplary antigen library of the present disclosure having all HLA allotypes and using peptides 8-11 amino acids in length (8mer-11 mer). In this example, yeast cells were electroporated with nucleic acids encoding an antigen library of HLA-antigen polypeptide complexes (pHLA library).

The electroporation method for expressing pHLA in yeast is as follows:

day 0:

1. three 2.5L baffled flasks and one 250mL baffled flask were autoclaved for expansion of the proliferating yeast culture.

2. A yeast peptone glucose medium (YPD) is prepared comprising bacterial peptone, glucose and yeast extract.

3. Two 5ml EBY100 yeast cultures were prepared and shaken overnight at 30 ℃.

4. Plasmid pYAL — 3T (10 μ g) digested with HindIII, NheI or NheI and BamHI restriction enzymes was prepared, and a plasmid containing SEQ ID NO: insert (50. mu.g) of the library of 210 to 411. The pYAL-3T vector (SEQ ID NO: 485) is a derivative of the pCT vector (SEQ ID NO: 486; Invitrogen), Table 8, and the maps are provided in FIGS. 3A and 3B. Tables 9 and 10 include the characteristics of pYAL — 3T and pCT, respectively. pYAL — 3T differs from pCT by at least: addition and ligation of linkers for human B2M as an orientation for the C-terminal displayed protein scaffold (Aga2) of the pHLA library. pYAL _3T (Gee2018 a) has been described.

Table 8: nucleotide sequences of pYAL _3T and pCT vectors

Table 9: characterization of the pYAL-3T vector

Table 10: characterization of the pCT vector

Feature(s)	Position of
		GAL1 promoter	5217-451
T7	475-494
		Aga2 leader sequence	534-587
Joint	588-632
		hB2m	633-929
Joint	930-989
		Epitope tag (cMyc)	990-1019
Joint	1020-1064
		Aga2	1065-1271
CEN-ARS pRS	2499-3009
		AmpR	3339-3998
LacO	5118-5140

Day 1: the two yeast cultures from day 0, step 3 were passaged and shaken overnight at 30 ℃ by adding 100 μ l of each of the two yeast cultures to 5ml of fresh YPD.

Day 2:

1. the Optical Density (OD) of the overnight culture from day 1 was measured.

2. In a 2.5L baffled flask, using YPD, 300ml of OD 0.3 yeast culture from step 1 was used to prepare a new culture.

3. Preparation of 3ml of 1M Tris pH 8.0/1M 1, 4-Dithiothreitol (DTT)

4. Preparation of 15ml of 2M lithium acetate (LiAc)/10mM Tris, 1mM EDTA (TE)

5. Cultures were propagated to an OD of 1.6-2.0.

6.3 ml of Tris/DTT was added.

7. 15ml of 2M LiAc/TE was added.

8. Cultures were propagated at 30 ℃ for 15 minutes while shaking at 225 revolutions per minute (rpm).

9. The cultures were centrifuged at 3000Xg for 3 minutes.

10. The pellet was resuspended in 50mL of cold E-buffer.

11. The suspension from step 10 was centrifuged at 3000Xg for 3 minutes at 4 ℃.

12. Steps 10 and 11 are repeated twice.

13. The residual buffer was removed.

14. The pellet was resuspended in 600. mu. L E-buffer.

15. Add 50. mu.g of insert and 10. mu.g of digested plasmid from step 4 on day 0, the total volume of buffer, insert, plasmid and yeast should be about 1 mL.

16. Aliquots of 150. mu.L of the suspension from step 15 into ice-cold 2mm gap electroporation cuvettes.

17. Each cuvette was electroporated at 2.5 kV. The time constant should be 3 to 4ms¹In the meantime.

18. Three cold YPDs were added in 1mL volumes, and then the total volume was increased to 200mL using YPD

19. The electroporated yeast was incubated at 225rpm for 1 hour at 30 ℃ in a 250mL baffled flask.

20. The culture was centrifuged at 3500xg for 3 minutes to form a yeast cell pellet, the supernatant was decanted, and the yeast cell pellet was resuspended in 10mL of SDCAA (glucose casein amino acid, which also includes a yeast nitrogen source without amino acids and ammonium sulfate, sodium citrate, and citric acid monohydrate, pH 4.5).

Day 2: determination of titre

1. 990 μ L of SDCAA was added to each of the four Eppendorf tubes.

2. 10 μ L from step 20 above was added to a tube containing 990 μ L of SDCAA.

3. Pipette 100. mu.L of 10⁴The solution was added to a tube containing only 990 μ L of SDCAA.

4. Pipette 100. mu.L of 10⁵The solution was added to a tube containing only 990 μ L of SDCAA.

5. Transfer and suck 10 of 100. mu.L⁶The solution was added to a tube containing only 990 μ L of SDCAA.

6. 100 μ L of each dilution from steps 2-5 was spread on separate SDCAA plates and incubated at 30 ℃ for 3 days. Colonies on the plates were counted to determine titer. According to step 2, the colonies counted represent library x10⁴The diversity of (a). According to step 3, the colonies counted represent library x10⁵The diversity of (a). According to step 4, the colonies counted represent library x10⁶The diversity of (a). According to step 5, the colonies counted represent library x10⁷The diversity of (a).

7. 490ml of pH 4.5SDCAA was added to the remaining cell suspension from step 20 and incubated overnight at 30 ℃.

Day 3: after 24 hours, the OD of passage from step 8 was measured. The OD should be at least 5. Cultures were passaged to an OD of 1 in a total volume of 500mL of SDCAA.

Day 4: cells were passaged to an OD of 1 in a total volume of 500mL of SDCAA.

Day 5: induction was performed in SGCAA (galactosamine amino acid, which also includes a yeast nitrogen source without amino acids and ammonium sulfate, sodium citrate and citric acid monohydrate, pH 4.5) 72 hours after step 18 from day 2.

The formula is as follows:

1) e-buffer, 500ml

-0.6g of Tris base,

-91.09g sorbitol (1M)

73.50mg CaCl2(1 mM; considered to make a 1M stock solution) in ddH2O to a final volume of 500ml, pH 7.5. Filtration through a 0.22 μm membrane.

2)1M Tris/1M DTT，3ml

0.462g of 1, 4-dithiothreitol in 3ml of 1M Tris, pH 8.0 and sterilized by filtration.

3)2M LiAc/TE solution, 15ml

1.98g LiAc in 10ml TE (10mM Tris, 1mM EDTA), sterilized by filtration.

Example 3: characterization of pHLA expression

This example describes the characterization of the expression of HLA-antigen polypeptide complexes on the electroporated yeast cells of example 2. These expression measurements included FACS analysis to determine the level of peptide-MHC displayed on the yeast cell surface and indicate the functionality of the random yeast display library. The expression of pHLA in yeast was characterized as follows:

material

1. Yeast library from example 2

PBSM (1 XPBS, 1g/L bovine serum albumin, EDTA, pH 7.4; filtered)

3. Anti-myc (FITC fluorophore conjugated) antibodies

4.96 hole U-shaped bottom plate

Optionally:

5. anti-V5 (647 fluorophore conjugated) antibody

6. anti-HA (BV421 fluorophore conjugated) antibodies

7. anti-VSV (PE fluorophore conjugated) antibodies

Cell preparation

1. The optical density of the yeast culture was measured on a NanoDrop. OD600 readings between 0 and 1 were taken at 1:20 culture: SDCAA dilutions induced in the linear range of cultures from 2 to 3 days at 20 ℃.

2. Samples of yeast cultures were transferred to wells of a 96-well plate. For yeast cultures with an OD600 of about 10, 25. mu.L of culture was used. Monochromatic and unstained controls were included.

3. To each well was added PBSM to a final volume of 200 μ Ι.

4. Centrifuge 96 well plates at 2500Xg for 2 minutes.

5. The supernatant was removed.

Staining cells

1. Each cell pellet was resuspended in 100. mu.l PBSM

2. Add 1. mu.l of antibody as appropriate.

3. Incubate at 4 ℃ for 30 minutes in the absence of light (e.g., in the dark).

Cells were washed and assayed for pHLA expression

1. Centrifuge 96 well plates at 2500Xg for 2 minutes.

2. The supernatant was removed.

3. Each pellet was resuspended in 200. mu.l PBSM.

4. Centrifuge 96 well plates at 2500Xg for 2 minutes.

5. The supernatant was removed.

6. Each pellet was resuspended in 200. mu.l PBSM.

7. Samples from each well were analyzed using CytoFlex.

As a result: expression of HLA-antigen polypeptide complexes (peptides of SEQ ID NOS: 8, 11, 14, 18, 21+24, 28, 32, 36, 40-44, 47, 50, 53, 56, 65, 75, 69, 77+80, 89, 95, 99, 102+106, 108, 111+114, 117+120, 124, and 125) was determined by flow cytometry and is shown in FIG. 4. Electroporated yeast cells were stained with antibodies targeting epitope tags expressed by HLA-antigen polypeptide complexes. FITC-A staining corresponds to HLA-antigen polypeptide complexes expressing the c-Myc tag. Antibody-epitope tag binding was used as an alternative for determining pHLA expression, as shown in figure 4, ranging from SEQ ID NO: 56 to SEQ ID NO: 26.3% expression of 177+ 180.

Example 4: functional validation of pHLA expression

This example describes the functional validation of pHLA expression on the electroporated yeast cells of example 2 with a candidate TCR. When the candidate TCRs are allotypically matched, the expected target antigen for pHLA can be identified from up to 6 libraries. The functional verification method for expression of pHLA in yeast is as follows:

HLA-antigen polypeptide sequence libraries, such as those disclosed herein, minimally express about 25% of the total antigen polypeptide sequence of a single 9mer length peptide presented by HLA-a1 (Gee2018b), and express less than about 5% of a single length peptide (e.g., 8mer) having a mixed length of peptides presented by HLA-a2 (e.g., 8mer, 9mer, 10mer, 11mer) (Gee2018 a). The isolated TCR of interest targets an 8mer antigen from an HLA-antigen polypeptide complex, although expression of a single length peptide of the HLA-antigen polypeptide sequence of the 8mer length peptide is less than about 5%. These isolated TCRs of interest were stimulated by one or more HLA-antigen polypeptide complexes in an in vitro co-culture assay (Gee2018 a; see figures 5C and 7A therein). Libraries of HLA-antigen polypeptides have been screened and peptides have been isolated that bind TCRs of known specificity (Gee2018 a). Although the minimum expression level necessary for a functional HLA-antigen polypeptide library of the present disclosure has not been determined, the data show that less than 15% expression can result in an HLA-antigen polypeptide library useful in the methods described herein.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

All publications, patent applications, issued patents, and other documents cited in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document were specifically and individually indicated to be incorporated by reference in its entirety. To the extent that a definition in this disclosure conflicts, the definition contained in the text incorporated by reference is excluded.

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