Engineered cell for developing neoantigen of mhc

Abstract

Disclosed is an engineered cell for developing a neoantigen of MHC, an engineered cell capable of displaying a trimer of a major histocompatibility complex and an antigen peptide, a nucleic acid encoding the trimer, and the use of the engineered cell and a preparation method therefor. The engineered cell can be used for accurate and efficient high-throughput screening of MHC-II target peptides and for the development of the neoantigen and a relevant vaccine and immunotherapy thereof. Also disclosed are an engineered cell capable of displaying a major histocompatibility complex, a nucleic acid encoding the MHC, and the use of the engineered cell and a preparation method therefor. The engineered cell can be used for accurate and efficient screening of MHC-II target peptides, and are applied to the development of a vaccine and T cell immunotherapy.

Description

Engineering cells to develop MHC neoantigens Technical Field

[0001] The present application relates to cell surface display technology, and in particular to an improved technical solution for displaying MHC-II and its trimer with antigenic peptides on the surface of yeast cells. Background Art

[0002] T cell-mediated immune responses play a crucial role in many diseases, including tumors, autoimmunity, transplant rejection, and infectious diseases. As central regulators of adaptive immunity, CD4+ T cells, activated by their receptors (TCRs), require specific recognition of antigens or pathogenic peptides bound and presented by major histocompatibility complex class II (MHC-II). MHC-II is a heterodimeric transmembrane protein composed of α and β chains, each containing two domains. These proteins capture antigenic peptides processed internally by professional antigen-presenting cells (APCs) and present them on the APC surface for recognition by CD4+ T cells, initiating downstream immune responses. The peptide-binding groove of MHC-II, formed by the α1 and β1 domains, contains several pocket-like structures that favor the specific side chains of "anchor" residues on the register of antigenic peptides. Therefore, the peptide register that MHC-II can accommodate has a certain degree of pan-activity, in other words, it can bind and present many different antigenic peptides, but the special convex and concave spatial structure of the anchor-pocket limits the types of peptides presented by a given MHC-II to a certain extent. Therefore, a deep understanding of the peptide binding function of MHC-II and then determining the residue preferences of various sites in the peptide register are crucial for the discovery of new tumor antigens, autoimmune regulation, vaccine design, infectious disease prevention, and transplant rejection relief.

[0003] The human MHC, or human leukocyte antigen (HLA), is a highly polymorphic, sugar-modified transmembrane protein known to occur naturally. This polymorphism is associated with numerous naturally occurring HLA haplotypes, subtypes (DR, DQ, DP), and thousands of alleles. Furthermore, the MHC protein expressed by each allele can bind and present a variety of peptide fragments. Therefore, studying the peptide-binding properties of various MHC-II proteins and defining the epitope maps of various peptide antigens is a highly challenging task, requiring high-throughput, high-content genetic protein engineering approaches. Conventional approaches involve purifying soluble recombinant MHC-II molecules from various expression systems (such as B cell lines, insect cells, yeast, or Escherichia coli) and analyzing their binding to various peptides. Other studies focus on evaluating peptide binding to MHC-II expressed on the surface of APCs. The peptide fragments used in these approaches are either chemically synthesized using solid-phase methods or generated using genetic methods such as phage display. This inefficient peptide fragment synthesis, labor-intensive soluble protein preparation, lengthy binding assays, or the generation of non-quantitative data (such as peptide abundance) limit the efficiency and yield of these methods for mapping specific binding epitope peptides across a large number of existing MHC-II alleles. Alternatively, eukaryotic cell surface display technology, which has the advantages of directed evolution, can be used to express specified MHC-II alleles to perform peptide binding assays on the cell surface. As a eukaryotic single-cell system, yeast has the advantages of simple molecular cloning and expression of eukaryotic post-translationally modified proteins, making it an ideal platform for developing this approach.

[0004] Summary of the Invention

[0005] This application provides engineered cells capable of displaying the major histocompatibility complex and its trimer with an antigenic peptide, nucleic acids encoding the complex and its trimer with an antigenic peptide, as well as uses of the engineered cells and methods for preparing the cells. The engineered cells can be used for accurate and efficient high-throughput screening of MHC-II target peptides and affinity analysis of MHC-II for target peptides.

[0006] Specifically, the present application relates to: 1. A cell comprising a major histocompatibility complex (MHC) and a single-chain domain, wherein the complex comprises an α chain and a β chain, wherein:

[0007] The α chain is linked to a first protein binding domain to form a first fusion protein,

[0008] The beta chain is linked to a second protein binding domain to form a second fusion protein,

[0009] The single-chain domain non-covalently binds to the α chain and the β chain, the first fusion protein binds to the second fusion protein to form the complex, and the complex and the single-chain domain form a trimer, wherein,

[0010] The first protein binding domain binds to the second protein binding domain to enhance or promote the formation of a trimer formed between the complex and the single-chain domain.

[0011] 2. A cell according to item 1, wherein the first protein binding domain is non-covalently bound to the second protein binding domain; or the first protein binding domain is covalently linked to the second protein binding domain, and the covalent bond is preferably a disulfide bond.

[0012] 3. The cell according to item 1, wherein the cell is a yeast cell.

[0013] 4. The cell according to item 1, wherein the single-chain domain is bound to the surface of the cell by covalent fusion with a molecule on the cell surface.

[0014] 5. The cell according to item 4, wherein the α chain or the β chain is bound to the molecule on the cell surface by non-covalent binding to the single-chain domain.

[0015] 6. The cell of item 4, wherein the molecule is a protein.

[0016] 7. The cell of item 6, wherein the protein is endogenous to the cell.

[0017] 8. The cell according to item 6, wherein the protein is Aga2p, α-lectin, α-lectin, Floccin, Cwplp, Cwp2p or Tip1p.

[0018] 9. The cell according to item 6, wherein the protein is Aga2p.

[0019] 10. The cell according to item 9, wherein the amino acid sequence of Aga2p is shown in SEQ ID NO: 1.

[0020] 11. A cell according to item 1, wherein the single-chain domain is a peptide segment of 9 to 30 amino acids in length, and the single-chain domain contains at least one continuous register peptide (register) of 9 amino acids in length that can be specifically recognized by the complex or a registered peptide variant (variant) that can be bound by the complex, preferably the register peptide or its variant is selected from the amino acid sequence shown in any one of SEQ ID NOs: 20-38.

[0021] 12. The cell according to item 11, wherein the amino acid sequence of the single-chain domain is as shown in any one of SEQ ID NOs: 2-6 or 46-77.

[0022] 13. The cell according to item 1, wherein the first protein binding domain is a first leucine zipper domain, and the second protein binding domain is a second leucine zipper domain, wherein the first leucine zipper domain and the second leucine zipper domain that form a complete leucine zipper can be used interchangeably in the first fusion protein and the second fusion protein; or

[0023] The first protein binding domain is an FcA domain, and the second protein binding domain is an FcB domain, wherein the FcA domain and the FcB domain that form the FcAB dimer can be used interchangeably in the first fusion protein and the second fusion protein.

[0024] 14. The cell according to item 13, wherein the amino acid sequence of the first leucine zipper domain is shown in SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10;

[0025] Correspondingly, the amino acid sequence of the second leucine zipper domain is shown in SEQ ID NO: 8 or SEQ ID NO: 7 or SEQ ID NO: 10 or SEQ ID NO: 9.

[0026] 15. The cell according to item 13, wherein the FcA domain and the FcB domain have the same or different amino acid sequences, preferably selected from any one of the amino acid sequences shown in SEQ ID NOs: 11-19;

[0027] Preferably, the Fc A domain and FcB domain are capable of increasing the expression display amount and / or copy number of the complex;

[0028] Preferably, the FcA domain comprises only the first CH3 domain, and the FcB domain comprises only the second CH3 domain; or

[0029] Preferably, the FcA domain comprises a first CH2 domain and a first CH3 domain, and the FcB domain comprises a second CH2 domain and a second CH3 domain;

[0030] It is further preferred that the first CH2 domain and the first CH3 domain of the first protein-binding domain are connected via a linker, and the second CH2 domain and the second CH3 domain of the second protein-binding domain are connected via a linker.

[0031] 16. The cell of item 15, wherein either or both of the FcA domain and the FcB domain comprises one or more amino acid modifications that achieve any one, two, or three of the following:

[0032] (1) enhance or stabilize the FcA domain and / or the FcB domain itself ("Fc5"),

[0033] (2) enhancing or stabilizing the covalent or non-covalent binding between the FcA domain and the FcB domain ("FcM"),

[0034] (3) Reducing or avoiding mispairing of the α chain and β chain caused by mispairing of the FcA domain with the FcB domain ("FcN").

[0035] 17. The cell according to any one of claims 1, wherein the first protein-binding domain is linked to the C-terminus of the α chain, and the second protein-binding domain is linked to the C-terminus of the β chain. Preferably, the signal peptide for promoting the expression of the first and second fusion proteins is SPP SP or AGA2 SP. More preferably, the amino acid sequence of AGA2 SP is shown in SEQ ID NO: 39, and the amino acid sequence of SPP SP is shown in SEQ ID NO: 45.

[0036] 18. The cell according to item 1, wherein the complex is an MHC class I molecule or an MHC class II molecule, preferably the complex is an HLA class I molecule or an HLA class II molecule.

[0037] 19. The cell according to item 18, wherein the α chain is encoded by HLA-DRA*01 or other alleles of the HLA-DRA family, and the β chain is encoded by HLA-DRB1*01 or HLA-DRB1*03 or HLA-DRB1*04 or HLA-DRB1*15 or other alleles of the HLA-DRB family; or

[0038] The α chain is encoded by HLA-DQA1*01 or HLA-DQA1*03 or HLA-DQA1*05 or other alleles of the HLA-DQA1 family, and the β chain is encoded by HLA-DQB1*02 or HLA-DQB1*03 or HLA-DQB1*05 or HLA-DQB1*06 or other alleles of the HLA-DQB1 family; or

[0039] The α chain is encoded by HLA-DPA1*01:03 or HLA-DPA1*02:02 or other alleles of the HLA-DPA1 family, and the β chain is encoded by HLA-DPB1*01:01 or HLA-DPB1*02:01 or HLA-DPB1*04:01 or HLA-DPB1*04:02 or other alleles of the HLA-DPB1 family.

[0040] 20. A nucleic acid encoding a major histocompatibility complex (MHC), wherein the complex comprises an α chain and a β chain, wherein the α chain is attached to a first protein-binding domain and the β chain is attached to a second protein-binding domain, wherein the α chain and the β chain are non-covalently bound to a single-chain domain, and the first protein-binding domain binds to the second protein-binding domain to enhance or promote formation of a trimer formed between the complex and the single-chain domain.

[0041] 21. The nucleic acid according to item 20, wherein the major histocompatibility complex encoded by the nucleic acid is the major histocompatibility complex (MHC) according to any one of items 1 to 19.

[0042] 22. A method for preparing a major histocompatibility complex (MHC), the method comprising:

[0043] Transform a cell with the nucleic acid of item 20 or 21 and a nucleic acid encoding a single-chain domain,

[0044] and culturing the cell under conditions where the complex involved in any one of items 1 to 19 is expressed.

[0045] 23. The method of item 22, wherein the cell is a yeast cell.

[0046] 24. A method for identifying a peptide that binds to a major histocompatibility complex (MHC), the method comprising:

[0047] i) displaying the single-chain domain library in a cell according to any one of items 1 to 19,

[0048] and ii) detecting trimers on the surface of monoclonal cells in a cell library displaying the single-chain domain involved in any one of items 1 to 19, thereby identifying peptides binding to the complex involved in any one of items 1 to 19.

[0049] 25. A method according to claim 24, wherein the single-chain domain comprises a peptide, or a mutant of a peptide, or a library of mutants of a peptide, or a mixture of peptides.

[0050] 26. A method according to item 24 or 25, wherein the peptide, or a mutant of a peptide, or a library of mutants of a peptide, or a mixture of peptides is non-covalently bound to the complex.

[0051] 27. A registered peptide or a variant thereof capable of binding to the major histocompatibility complex (MHC), wherein the registered peptide or the variant thereof is selected from the amino acid sequence shown in any one of SEQ ID NOs: 20-38.

[0052] The present application also relates to an engineered cell capable of displaying the major histocompatibility complex (MHC), a nucleic acid encoding the MHC, and uses and methods for preparing the engineered cell. The engineered cell improves the sensitivity and accuracy of determining the affinity between a target peptide and an MHC-II protein displayed by yeast, while also enabling short-term, efficient, and high-throughput assays based on competitive binding between test and reference peptides and the MHC-II protein displayed by yeast.

[0053] The present application also relates to: 28. A cell comprising a major histocompatibility complex (MHC), wherein the complex comprises an α chain and a β chain, wherein,

[0054] The α chain is connected to the first protein binding domain to form a first fusion protein,

[0055] The β chain is connected to a second protein binding domain to form a second fusion protein,

[0056] The first fusion protein binds to the second fusion protein to form the complex,

[0057] The first protein binding domain and the second protein binding domain enhance the binding of the α chain and the β chain to form the MHC.

[0058] 29. The cell of item 28, wherein the cell is a yeast cell.

[0059] 30. A cell according to claim 28, wherein the complex is bound to the surface of the cell by linking the α chain or the β chain to a molecule on the surface of the cell.

[0060] 31. A cell according to claim 30, wherein the additional α chain or the additional β chain in the complex is bound to the molecule on the cell surface by non-covalently binding to the β chain or the α chain.

[0061] 32. The cell of item 30, wherein the molecule is a protein.

[0062] 33. A cell according to item 32, wherein the protein is endogenous to the cell.

[0063] 34. The cell according to item 32, wherein the protein is Aga2p, α-lectin, α-lectin, flocculin, Cwp1p, Cwp2p or Tip1p.

[0064] 35. The cell of item 32, wherein the protein is Aga2p.

[0065] 36. The cell according to item 35, wherein the amino acid sequence of Aga2p is as shown in SEQ ID NO: 1.

[0066] 37. The cell of item 28, wherein the first protein binding domain non-covalently binds to the second protein binding domain; or

[0067] The first protein binding domain is covalently bound to the second protein binding domain, and preferably the covalent binding is through a disulfide bond.

[0068] 38. The cell according to item 28, wherein

[0069] The first protein binding domain and the second protein binding domain form an FcAB domain;

[0070] Preferably, the first protein binding domain is an FcA domain or an FcB domain, and the second protein binding domain is an FcB domain or an FcA domain, wherein,

[0071] It is further preferred that the FcA domain and FcB domain can increase the expression level and / or copy number of the complex.

[0072] 39. The cell according to item 38, wherein

[0073] The FcA domain only comprises the first CH3 domain, and the FcB domain only comprises the second CH3 domain; or

[0074] The FcA domain comprises both a first CH2 domain and a first CH3 domain, and the FcB domain comprises both a second CH2 domain and a second CH3 domain;

[0075] It is further preferred that the first CH2 domain and the first CH3 domain of the first protein-binding domain are connected via a linker, and the second CH2 domain and the second CH3 domain of the second protein-binding domain are connected via a linker.

[0076] 40. The cell according to item 39, wherein

[0077] The FcA domain and the FcB domain are selected from any one of the amino acid sequences shown in SEQ ID NOs: 11-19, or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 11-19.

[0078] 41. The cell of item 39 or 40, wherein either or both of the FcA domain and the FcB domain comprise one or more amino acid modifications,

[0079] The modification can achieve any of the following:

[0080] enhance or stabilize the FcA domain and / or the FcB domain itself ("Fc5"),

[0081] enhancing or stabilizing the covalent or non-covalent binding between the FcA domain and the FcB domain ("FcM"),

[0082] Mispairing of the α and β chains due to mispairing of the FcA domain with the FcB domain is reduced or avoided ("FcN").

[0083] 42. The cell according to any one of items 28 to 41, wherein the first protein binding domain is linked to the C-terminus of the α chain, and the second protein binding domain is linked to the C-terminus of the β chain. Preferably, the signal peptide for promoting the expression of the first fusion protein and the second fusion protein is SPP SP or AGA2 SP, and further preferably, the amino acid sequence of the AGA2 SP is as shown in SEQ ID NO: 39, and the amino acid sequence of the SPP SP is as shown in SEQ ID NO: 45.

[0084] 43. The cell according to any one of items 28 to 42, wherein the complex is an MHC class I molecule or an MHC class II molecule, preferably the complex is an HLA class I molecule or an HLA class II molecule.

[0085] 44. The cell according to item 43, wherein

[0086] The α chain is encoded by HLA-DRA*01 or other alleles of the HLA-DRA family, and the β chain is encoded by HLA-DRB1*01 or HLA-DRB1*03 or HLA-DRB1*04 or HLA-DRB1*15 or other alleles of the HLA-DRB family; or

[0087] The α chain is encoded by HLA-DQA1*01 or HLA-DQA1*03 or HLA-DQA1*05 or other alleles of the HLA-DQA1 family, and the β chain is encoded by HLA-DQB1*02 or HLA-DQB1*03 or HLA-DQB1*05 or HLA-DQB1*06 or other alleles of the HLA-DQB1 family; or

[0088] The α chain is encoded by HLA-DPA1*01:03 or HLA-DPA1*02:02 or other alleles of the HLA-DPA1 family, and the β chain is encoded by HLA-DPB1*01:01 or HLA-DPB1*02:01 or HLA-DPB1*04:01 or HLA-DPB1*04:02 or other alleles of the HLA-DPB1 family.

[0089] 45. A nucleic acid encoding a major histocompatibility complex (MHC), wherein the complex comprises an α chain and a β chain, wherein the α chain is linked to a first protein binding domain, and the β chain is linked to a second protein binding domain, wherein the first protein binding domain and the second protein binding domain are capable of binding to form the complex, and the first protein binding domain and the second protein binding domain form an FcAB dimer to enhance the binding of the α chain and the β chain to form the MHC.

[0090] 46. ​​The nucleic acid according to item 45, wherein the encoded major histocompatibility complex is the major histocompatibility complex (MHC) according to any one of items 1 to 17.

[0091] 47. A method for preparing a major histocompatibility complex (MHC), wherein the method comprises:

[0092] Transforming a cell with the nucleic acid of item 45 or 46,

[0093] and culturing the cell under conditions where the complex involved in any one of items 28 to 44 is expressed.

[0094] 48. The method of item 47, wherein the cell is a yeast cell.

[0095] 49. A method for identifying a peptide that binds to a major histocompatibility complex (MHC), wherein the method comprises:

[0096] i) contacting the cell of any one of items 28 to 44 with a peptide,

[0097] and ii) detecting the binding of the peptide to the complex according to any one of items 28 to 44, thereby identifying the peptide binding to the complex.

[0098] 50. The method of claim 49, wherein the peptide comprises a mixture of peptides.

[0099] 51. A method according to item 49 or 50, wherein the peptide competes with a reference peptide for binding to the complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1. CODAAH system design scheme. The peptide fused to the second subunit of the α-agglutinin protein (Aga2p) is fused to the N-terminus and C-terminus with HA and V5 epitope tags, respectively. Immunofluorescence staining with antibodies specific for either tag (e.g., anti-HA antibody markers) allows for detection of peptide expression and cell surface display levels. The extracellular domains of MHC-IIα and MHC-IIβ chains with C-terminal dimerization modifications are expressed using a separate vector independent of the peptide display vector. The peptide is fused to a yeast-secreted α-agglutinin protein (composed of Aga1p and Aga2p subunits, each formed by two -SH residues forming an -SS- structure) using classic yeast display methods and anchored to the cell surface. The MHC-IIα / β heterodimer enhances complexation efficiency through enhanced dimerization at the C-terminus and is anchored to the cell surface through non-covalent binding to the specific antigenic peptide, achieving peptide / MHC-IIα / β trimer co-display in yeast. The relative fluorescence levels of different fluorophores conjugated to anti-tag and anti-MHC-II (Anti-MHC-II) reagents indicate the degree of saturation of bound MHC-II pairs with specific peptide binding. EBY00: parental yeast strain for yeast display. GPI: glycosylphosphatidylinositol transports endogenous yeast proteins, which are translated and expressed from the yeast genome.

[0101] Figure 2 shows a schematic diagram of the target gene region of a selectable plasmid, which is the DR1 allele (HLA-DRA*01:01 / DRB1*01:01). Using this selectable plasmid, yeast can translate and express the extracellular domain of the MHC-II complex. The C-terminus of MHC-II is modified with a leucine tract (LZA or LZB) to enhance dimerization. Each expression cassette contains the GA1 or GAL10 promoter in the illustrated orientation, a fusion protein of HLA-II with a C-terminal modified strand, and an MFα terminator.

[0102] FIG3 is similar to FIG2 and shows a schematic diagram of the target gene region of the selectable plasmid, wherein the target gene is the DR4 allele (HLA-DRA*01:01 / DRB1*04:01).

[0103] FIG4 is similar to FIG2 and shows a schematic diagram of the target gene region of the selectable plasmid, wherein the target gene is the DQ6 allele (HLA-DQA1*01:02 / DQB1*06:02).

[0104] Figure 5 is a schematic diagram of the target gene region of the selectable plasmid, similar to Figure 2, wherein the target gene is the DR1 allele (HLA-DRA*01:01 / DRB1*01:01). The C-terminus of MHC-II is modified by an antibody crystallizable fragment (FcA or FcB) to enhance dimerization coupling.

[0105] FIG6 is similar to FIG5 and shows a schematic diagram of the target gene region of the selectable plasmid, wherein the target gene is the DR4 allele (HLA-DRA*01:01 / DRB1*04:01).

[0106] FIG7 is similar to FIG5 and shows a schematic diagram of the target gene region of the selectable plasmid, wherein the target gene is the DQ6 allele (HLA-DQA1*01:02 / DQB1*06:02).

[0107] Figure 8 shows immunofluorescence labeling and flow cytometry analysis of surface DR or DQ protein conformations in four yeast strains: secreting only HLA-II (background control), displaying LZ modification (labeled HLA-II-LZ), displaying FcAB modification (labeled HLA-II-Fc), and displaying FcAB modification with an AGA2 SP signal peptide for enhanced extracellular delivery (labeled HLA-II-Fc (w / AGA2 SP)). Flow cytometry results are displayed as histograms. The median fluorescence intensity (MFI) of the DR or DQ protein under each condition is shown. The level of HLA-II-LZ or HLA-II-Fc display in yeast can be quantified using the signal-to-noise difference (ΔMFI = MFI_LZ - MFI_BC or MFI_Fc - MFI_BC).

[0108] Figure 9 shows the difference between the control group that only secretes DR1 (no pep+DR background control) and the control group that only displays DR specific peptide (+HA 308-316 no DR control), showing HA 308-316 The peptide secretes LZ to modify DR1-LZ, which displays HA 308-316 The peptide also secretes Fc-modified DR1-LZ with AGA2 SP signal peptide to enhance extracellular delivery, which not only displays HA 308-316 The peptide secretes LZ to modify DR4-LZ, which displays HA 308-316The peptide also secretes Fc-modified DR4-LZ with an AGA2 SP signal peptide for enhanced extracellular delivery. Six yeast strains were co-labeled for surface HA tag and DR protein conformation using immunofluorescence and flow cytometry. Flow cytometry results are displayed as dot plots (SSC scatter vs. HA tag) or histograms. The dot plots indicate the percentage of gated HA+ positive cell subpopulations, while the histograms indicate the median fluorescence intensity (MFI) of DRα / β proteins in the HA+ subpopulations. DR display levels can be quantitatively analyzed as shown in Figure 8.

[0109] Figure 10 shows both HA 308-316 The parent yeast library (+pep) in which the peptide variants also secrete DR1 was immunofluorescently co-labeled for surface HA tag and DR protein conformation and flow cytometrically sorted. HA+ / DR+ double-positive cells from four rounds of sorting (S1, S2, S3, and S4) and the HA- / DR- double-negative cells that secrete only DR1 (no pep) used as the background control in each round are shown in the HA tag vs. DR protein dot plots. Additionally, the SSC side scatter vs. HA tag dot plots indicate the percentage of gated HA+ positive cell subpopulations, and the histogram plots indicate the median fluorescence intensity (MFI) of DRα / β proteins in the HA+ subpopulations. The increasing levels of DR display in each round can be quantitatively analyzed as shown in Figure 8.

[0110] Figure 11 shows both HA 308-316 Ten single clones selected from the peptide variant DR1-secreting yeast sublibrary were subjected to yeast PCR and target peptide base sequencing. The results were compared with the library DNA primers and the wild-type gene and peptide amino acid sequences. With the exception of clones #1 and #4, the sequencing peaks showed significant overlap, indicating that a single yeast cell can accommodate multiple recombinant plasmids transformed with them. Therefore, the fact that the dominant codon DNA and products corresponding to the dominant codons of the peptide variants in clones #5 and #7 are identical does not guarantee that the peptide variant-displaying recombinant plasmids generated from these two yeast clones are identical.

[0111] Figure 12 shows the extraction of yeast plasmids, transformation of E. coli, and subsequent extraction of one to three E. coli plasmids from each yeast strain and sequencing of target peptide bases for the four selected monoclonal clones (#1, #7, #9, and #10). The results were compared with the wild-type gene and its peptide amino acid sequence. The selected peptide display plasmids were further transformed into yeast to construct a peptide displaying HA. 308-316Peptide variants were also expressed in strains secreting DR1, and surface HA tags and DR protein conformation were co-labeled by immunofluorescence and analyzed by flow cytometry. Flow cytometry results are displayed as dot plots (SSC scatter plot vs. HA tag) or histograms. Dot plots indicate the percentage of gated HA+ positive cell subpopulations, while histograms indicate the median fluorescence intensity (MFI) of DRα / β proteins in the HA+ subpopulations. DR display levels can be quantitatively analyzed as shown in Figure 8.

[0112] FIG13 uses the display plasmid corresponding to the peptide numbered #1 to further transform yeast to construct a strain that displays both the #1 peptide and secretes DR4, and performs immunofluorescence co-labeling and flow cytometry of the surface HA tag and DR protein conformation. The flow cytometry results are displayed as dot plots (SSC side scatter vs HA tag) or histograms. Among them, the dot plots are annotated with the percentage of gated HA+ positive cell subpopulations, and the histograms are annotated with the median fluorescence intensity (MFI) of the HA+ subpopulation DRα / β protein. The DR display level can be quantitatively analyzed as shown in FIG8.

[0113] Figure 14. EBY100 yeast expressing soluble HLA-II (background control, or BC), displaying LZ-modified HLA-II (W / LZ), or displaying FcAB-modified HLA-II (W / Fc) were immunofluorescently labeled and flow cytometry analyzed for surface DR or DQ protein levels. Flow cytometry results are displayed as histograms. The median fluorescence intensity of the HLA-II α / β alleles and DR or DQ proteins under each condition is shown. The level of HLA-II-LZ or HLA-II-Fc yeast expression can be quantified using the signal-to-noise difference (ΔMFI = MFI_LZ - MFI_BC or MFI_Fc - MFI_BC).

[0114] Figure 15. EBY100 yeast expressing soluble HLA-II (BC), displaying LZ-modified HLA-II (W / LZ), or displaying FcAB-modified HLA-II with AGA2 signal peptide-directed extracellular delivery (W / Fc) were subjected to immunofluorescence labeling and flow cytometry analysis of surface DR protein levels. Flow cytometry results are displayed as histograms. The median DR protein fluorescence intensity for each DRα / β allele and condition is shown. DR-LZ or DR-Fc yeast display levels can be quantified using the signal-to-noise difference (ΔMFI = MFI_LZ - MFI_BC or MFI_Fc - MFI_BC).

[0115] Figure 16 EBY100 yeast cells displaying LZ-modified DR1 (DR-LZ) or displaying FcAB-modified DR1 (DR1-Fc) guided by AGA2 signal peptide for extracellular delivery, and immunofluorescence co-labeling and flow cytometry analysis of surface DR protein levels and indicator peptide levels. Flow cytometry results are displayed as dot-plots or histograms. 306-318 ) or negative control peptide (Bio-aI). Cell population gated analysis and median DR protein fluorescence intensity (for analysis of a DR-positive yeast subpopulation) are shown in the figure. Biotin levels of the biotinylated indicator peptide bound to the yeast surface can be quantified by the signal-to-noise difference (ΔMFI = MFI_Bio-pep - MFI_no peptide).

[0116] Figure 17 EBY100 yeast cells displaying LZ-modified DR1 (DR-LZ) or displaying FcAB-modified DR1 (DR1-Fc) guided by AGA2 signal peptide extracellular delivery, and with different concentrations of biotinylated indicator peptide (Bio-HA 306-318 ) were bound and immunofluorescence co-labeling and flow cytometry were performed to measure surface DR protein and indicator peptide levels. Flow cytometric analysis results are shown in Figure 16 , followed by single-site binding kinetic curve fitting. The apparent equilibrium dissociation constant, KD,app, calculated from the fitted curve is shown in the figure.

[0117] Figure 18 EBY100 yeast cells displaying LZ-modified DR1 (DR-LZ) or displaying FcAB-modified DR1 (DR1-Fc) guided by AGA2 signal peptide for extracellular delivery, and immunofluorescence co-labeling and flow cytometry analysis of surface DR protein levels and indicator peptide levels. Flow cytometry results are displayed in Dot-plot or histogram format. 306-318 ) and minus (no competitor) plus excess positive competitive peptide (10 times HA 306-318 ) or negative competitor peptide (10x aI). Gated cell populations and median DR protein fluorescence intensity (for analysis of a DR-positive yeast subpopulation) are shown in the figure. The level of biotinylation of the biotinylated indicator peptide bound to the yeast surface can be quantified by the signal-to-noise difference (ΔMFI = MFI_Bio-pep-MFI_no indicator).

[0118] Figure 19 EBY100 yeast cells displaying LZ-modified DR1 (DR-LZ) or displaying FcAB-modified DR1 (DR1-Fc) guided by AGA2 signal peptide for extracellular delivery, and 10 μM biotinylated indicator peptide (Bio-HA 306-318 ) and different concentrations of competitive peptide (HA 306-318 ) were combined with immunofluorescence co-labeling and flow cytometry to determine surface DR protein and indicator peptide levels. The flow cytometric results were analyzed as shown in Figure 18 , followed by single-site competition binding kinetic curve fitting. The IC50 values ​​calculated from the fitted curves are shown in the figure. Specific implementation plan

[0119] This application provides a scheme for co-expressing an antigen peptide gene and an MHC-II allele in yeast, and co-displaying the expressed protein product, namely the peptide / MHC-II trimer, on the yeast surface. This scheme not only enables yeast to co-express complex MHC-II alleles, but also solves the problem of low co-display of peptide / MHC-II trimers. It greatly improves the efficiency of the development of new antigens related to MHC-II / antigen peptides, and at the same time, provides new ideas for the development of various vaccines, including oral vaccines; it also provides solutions for the development of targets for precision immunotherapy.

[0120] Specifically, the scheme of the present application uses a yeast co-display system (Figure 1), which is named CODAAH (CO-Display of Antigen-ligand and Assicated HLA-II). In order to co-display the peptide fragments and the MHC-II complex as a trimer on the cell surface, the present application first connects the protein product of the peptide fragment gene to the yeast surface by genetic fusion. Since the peptide fragments come from T cell antigens, the MHC-II complex secreted in a soluble form can non-covalently bind to the peptide fragments displayed by yeast, thereby realizing the co-display of the peptide / MHC-II trimer on the yeast surface. By modifying the C-termini of these MHC-II proteins, including fusing leucine zipper structure fragments or fusing antibody heavy chain crystallizable fragments, the display amount of peptide / MHC-II trimers on the surface of Saccharomyces cerevisiae is significantly improved.

[0121] definition

[0122] Although various embodiments and aspects of the present invention are shown and described herein, it will be apparent to those skilled in the art that these embodiments and aspects are provided merely as examples. Without departing from the present invention, those skilled in the art will now appreciate that many variations, changes, and replacements may be employed. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in implementing the present invention.

[0123] As used herein, percentages of "identity," such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identity, refer to a degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. For example, the percentage of positions with identical bases or amino acid residues is determined as a ratio of the total number of positions after two sequences have been aligned to have identical residues at as many positions as possible, such as by introducing gaps. Percentages of "identity" can be determined using software programs known in the art. Preferably, the alignment is performed using default parameters. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found on the Internet at the following address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0124] As used herein, the "complementarity" of nucleic acids refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with another nucleic acid molecule (e.g., about 5, 6, 7, 8, 9, 10 out of 10 are about 50%, 60%, 70%, 80%, 90% and 100% complementary, respectively). "Completely complementary" refers to that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. For single bases or nucleotides, according to the Watson-Crick base pairing principle, when A pairs with T or U, or C pairs with G or I, they are considered complementary or matching, and vice versa; all other base pairings are considered non-complementary. In this application, the "complementary polynucleotide sequence" of a polynucleotide sequence refers to a polynucleotide sequence that is completely complementary to the polynucleotide sequence.

[0125] As used herein, a "conservative substitution variant" of a protein, polypeptide or amino acid sequence refers to one in which one or more amino acid residues undergo amino acid substitution without changing the overall conformation and function of the protein or enzyme, including but not limited to replacing the amino acids in the amino acid sequence of the parent protein in the manner described by the aforementioned "conservative substitution". Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different. For example, a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. "Conservative substitution variants" also include polypeptides or enzymes with more than 60% amino acid identity as determined by the BLAST or FASTA algorithm, preferably more than 75%, preferably more than 85%, and even more than 90%, and have the same or substantially similar properties or functions as the natural or parent protein or enzyme.

[0126] In this article, "coding" refers to i) the genetic information contained in the DNA sequence that can be transcribed into an RNA molecule, and / or ii) the genetic information contained in the RNA molecule that can be translated into an amino acid sequence. Therefore, as used herein, "coding sequence" can be used to refer to a ribonucleotide (RNA) sequence or a fragment thereof in an mRNA precursor or mature mRNA that can be translated into a protein, and can also refer to the complementary sequence of a deoxyribonucleotide (DNA) sequence or a fragment thereof that is used as a template for transcribing the mRNA precursor or mature mRNA. In addition, the "coding sequence" of the present application can further include polynucleotide sequences that encode proteins, functional nucleic acids, or fragments thereof, such as miRNA, shRNA, dsRNA, guide RNA, Poly (A) tail, 5'UTR, 3'UTR, etc. Among them, a DNA molecule containing genetic information that can be transcribed into an RNA molecule is called the "coding nucleic acid" of the RNA molecule; an RNA molecule containing genetic information that can be translated into an amino acid sequence is called the "coding nucleic acid" of the amino acid sequence.

[0127] As used herein, the term "MHC-II" or "MHC-II complex" refers to the major histocompatibility class II (MHC-II) complex molecules typically found on antigen presenting cells (including dendritic cells, B cells, and phagocytes). The MHC-II complex helps, for example, regulate the immune system by presenting antigenic peptides. The MHC-II complex includes two human leukocyte antigen (HLA) proteins, referred to herein as "alpha chain" or "α chain" and "beta chain" or "β chain," which combine to form a heterodimer. The α1 and β1 regions of the α and β chains are respectively close to and aggregate together to form a peptide binding domain. The α2 and β2 regions of the α and β chains are respectively located closer to the cell membrane and form immunoglobulin-like domains. The N-terminal regions of the α and β chains include the α1 and β1 regions, and the C-terminal regions of the α and β chains include the α2 and β2 regions. The α and β chains are typically connected to the cell surface through a transmembrane domain.

[0128] The term "human leukocyte antigen" or "HLA" refers to the group of proteins encoded by the human major histocompatibility complex (MHC) gene complex. The HLA genes that encode the HLA protein group have different alleles, which provide different functions for the gene products. HLA proteins belonging to the MHC-II class (e.g., HLA-DP, HLA-DQ, HLA-DR) generally present peptides generated by antigens from outside the cell. MHC-II class proteins include HLA-DP, HLA-DQ, and HLA-DR, which are heterodimeric cell surface receptors consisting of α and β chains.

[0129] As provided herein, the terms "HLA-DR1, DR4, DR15 alpha chain" or "HLA-DR1, DR4, DR15 alpha chain protein" include any recombinant or naturally occurring form of the human leukocyte antigen (HLA) HLA-DR alpha chain protein, also known as MHC class II HLA-DRA, or variants or homologs thereof that retain HLA-DR alpha chain protein activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to HLA-DRA*01:01). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring HLA-DR4 alpha chain polypeptide. In an embodiment, the HLA-DR alpha chain is the protein identified by UniProt sequence reference P01903, a homologue or a functional fragment thereof.

[0130] The terms "HLA-DR β chain" or "HLA-DR β chain protein" as provided herein include any recombinant or naturally occurring form of the human leukocyte antigen (HLA) HLA-DR β chain protein, which can be a protein expressed by MHC-II class HLA-DRB1*01:01 or HLA-DRB1*04:01 or HLA-DRB1*15:01 or other alleles of the HLA-DRB family, or a variant or homolog thereof, which retains HLA-DR1 or DR4 or DR15 or other DR β chain protein activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the activity compared to the β chain of the protein expressed by HLA-DRB1*01:01 or HLA-DRB1*04:01 or HLA-DRB1*15:01 or other alleles of the HLA-DRB family). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring HLA-DR4 β chain polypeptide. In embodiments, the HLA-DR4 β chain is a protein identified by UniProt sequence reference P13762, a homolog, or a functional fragment thereof.

[0131] As provided herein, the terms "HLA-DQ6 α chain" or "HLA-DQ6 α chain protein" include any recombinant or naturally occurring form of the human leukocyte antigen (HLA) DQ α 1 chain (HLA-DQ6 α chain), also known as DC-1 α chain, DC-α, HLA-DCA, MHC class II DQA1, or variants or homologs thereof that retain HLA-DQ6 α chain protein activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to HLA-DQ6 α chain). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring HLA-DQ6 α chain polypeptide. In an embodiment, the HLA-DQ6 alpha chain is the protein identified by UniProt sequence reference P01909, a homolog or a functional fragment thereof.

[0132] As provided herein, the terms "HLA-DQ6 beta chain" or "HLA-DQ6 beta chain protein" include any recombinant or naturally occurring form of the human leukocyte antigen (HLA) DRB1 beta chain 1 protein (HLA-DQ6 beta chain), also known as MHC class II DQB1, HLA-DQB1, or variants or homologs thereof that retain HLA-DQ6 beta chain protein activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to HLA-DQ6 beta chain). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring HLA-DQ6 beta chain polypeptide. In an embodiment, the HLA-DQ6 beta chain is the protein identified by UniProt sequence reference P01920, a homolog or a functional fragment thereof.

[0133] For specific proteins described herein, the designated proteins include naturally occurring forms, variants, or homologs of any protein that retain protein transcription factor activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the native protein). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 consecutive amino acid portion) compared to the naturally occurring form. In other embodiments, the protein is a protein identified by its NCBI sequence reference. In other embodiments, the protein is a protein identified by its NCBI sequence reference, a homolog thereof, or a functional fragment thereof.

[0134] "Contacting" is used in accordance with its simple common meaning and refers to a process in which at least two different substances (e.g., a peptide and an MHC-II complex) are allowed to become sufficiently close to react, interact, or physically contact. It should be understood that the resulting reaction product can be produced directly by the reaction between the added reagents, or by an intermediate of one or more added reagents, which can be produced in the reaction mixture. The term "contacting" may include allowing two substances to react, interact, or physically contact, wherein the two substances may be, for example, nucleic acids and cells as provided herein. In embodiments, contacting includes, for example, allowing nucleic acids as described herein to enter cells.

[0135] As used herein, "cell" refers to a cell that performs metabolic or other functions sufficient to preserve or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a specific dye, or the ability to produce progeny. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, such as mammals, insects (e.g., Spodoptera exigua) and human cells.

[0136] When referring to, for example, a cell, nucleic acid, protein or vector, the term "recombinant" means that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or an alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. For example, a recombinant protein is a protein produced by a recombinant nucleic acid molecule. A nucleic acid molecule can include genetic material from a variety of sources, thereby including non-naturally occurring sequences. Recombinant DNA can be produced by methods known in the art of molecular biology or by synthetic methods. Thus, for example, recombinant cells express genes not found in the natural (non-recombinant) form of the cell, or express native genes that are abnormally expressed, underexpressed or not expressed at all. Transgenic cells and plants are cells and plants that express heterologous genes or coding sequences, usually as a result of recombinant methods.

[0137] The term "heterologous," when used to refer to portions of a nucleic acid, indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are often recombinantly produced, having two or more sequences from unrelated genes that are arranged to produce a new functional nucleic acid, such as a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0138] The term "exogenous" refers to a molecule or substance (e.g., a compound, nucleic acid, or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that is not derived from the cell or organism in which it is expressed. In contrast, the term "endogenous" or "endogenous promoter" refers to a molecule or substance that is native to or originates from a given cell or organism. For example, a yeast cell endogenous protein refers to a protein that is naturally expressed by the yeast cell. A nucleic acid encoding an endogenous protein can be introduced into a cell, thereby allowing the protein to be expressed. For example, a nucleic acid encoding Aga2p can be introduced into a yeast cell, thereby expressing Aga2p.

[0139] The term "expression" includes any step involved in the production of the polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion. Expression can be detected using conventional techniques for detecting proteins (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0140] "Biological sample" or "sample" refers to material obtained or derived from a subject or patient. Biological samples include tissue sections, such as biopsy and autopsy samples, and frozen sections for histological purposes. Such samples include body fluids, such as blood and blood components or products (e.g., serum, plasma, platelets, red blood cells, etc.), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells), feces, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. Biological samples are typically obtained from eukaryotic organisms, such as mammals, such as primates, such as chimpanzees or humans; cattle; dogs; cats; rodents, such as guinea pigs, rats, mice; rabbits; or birds; reptiles; or fish.

[0141] "Control" or "standard control" refers to a sample, measurement or value used as a reference, usually a known reference, for comparison with a test sample, measurement or value. For example, a test sample can be obtained from a patient suspected of having a given disease and compared with a known normal (unaffected) individual (e.g., a standard control subject). A standard control can also represent the average measurement or value collected from a group of healthy individuals without a given disease (i.e., a standard control population), such as a group of healthy individuals with a similar medical background, the same age, and weight. Standard control values ​​can also be obtained from the same individual, such as a patient sample obtained earlier before the onset of the disease. For example, a control can be designed to compare the therapeutic benefits based on pharmacological data (e.g., half-life) or treatment measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if the value of a given parameter varies greatly in the control, the change in the test sample will not be considered significant. Those skilled in the art will recognize that standard controls can be designed to assess any number of parameters (e.g., RNA levels, protein levels, specific cell types, specific body fluids, specific tissues, etc.).

[0142] Those skilled in the art will understand which standard controls are most appropriate in a given situation and will be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g., statistical significance) of data. For example, if the value of a given parameter varies greatly in the standard control, the change in the test sample will not be considered significant.

[0143] It should be understood that the present application includes various aspects, embodiments and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Other aspects, improvements and modifications within the scope of the present application will be apparent to those skilled in the art. Therefore, those of ordinary skill in the art will recognize that the scope of the present application also includes the improvements and modifications to the aspects and embodiments.

[0144] cell

[0145] In one aspect, the present application provides an engineered cell comprising a major histocompatibility complex (MHC) and a single-chain domain, wherein the complex comprises an α chain and a β chain, wherein:

[0146] The α chain is linked to a first protein binding domain to form a first fusion protein,

[0147] The beta chain is linked to a second protein binding domain to form a second fusion protein,

[0148] The single-chain domain non-covalently binds to the α chain and the β chain, and the first fusion protein binds to the second fusion protein to promote the α chain and the β chain to form the complex, so that the complex and the single-chain domain together constitute a trimer, wherein the first protein binding domain binds to the second protein binding domain to enhance or promote the formation of the trimer formed between the complex and the single-chain domain.

[0149] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a cell derived from a unicellular organism. In some embodiments, the cell is derived from a mononuclear eukaryotic organism. In some embodiments, the cell is a yeast cell. In some embodiments, the yeast is Saccharomyces cerevisiae. In some embodiments, the yeast is strain EBY100 (Boder & Wittrup, Yeast surface display for screening combinatorial polypeptide libraries, 1997).

[0150] In some embodiments, the single-chain domain is bound to the surface of the cell by covalent fusion with the molecule on the cell surface. In some embodiments, the single-chain domain is bound to the surface of the cell by a peptide bond with the molecule on the cell surface. In some embodiments, the single-chain domain is bound to the surface of the cell by a peptide chain with the molecule on the cell surface. Thus, in some embodiments, the α chain or the β chain is bound to the molecule on the cell surface by non-covalent binding with the single-chain domain. In some embodiments, the α chain and the β chain are bound to the molecule on the cell surface by non-covalent binding with the single-chain domain. In some embodiments, the molecule comprises a structure that can anchor the yeast cell wall. In some embodiments, the molecule comprises a structure that can anchor the yeast cell wall and a secretion signal peptide (or secretion signal region). In some embodiments, the structure that can anchor the yeast cell wall can directly or indirectly bind to cell wall glucan or cell wall mannan. In some embodiments, the yeast cell wall-anchorable structure can be covalently or non-covalently bound directly or indirectly to a cell wall glucan or a cell wall mannan. In some embodiments, the yeast cell wall-anchorable structure can be covalently or non-covalently bound directly or indirectly to a cell wall glucan or non-covalently bound to a cell wall mannan. In some embodiments, the yeast cell wall-anchorable structure and / or secretion signal peptide are derived from the α-lectin system and / or the floccin system (glycosylphosphatidylinositol anchor system and floccin domain anchor system). In some embodiments, the yeast cell wall-anchorable structure is directly or indirectly bound to or linked to a GPI anchor attachment signal region or a flocculation functional region. In some embodiments, the molecule is endogenous to the cell. In some embodiments, the molecule is selected from: Aga2p, α-lectin, α-lectin, floccin, Cwp1p, Cwp2p, or Tip1p. In some embodiments, the protein is Aga2p. In some embodiments, Aga2p is a protein encoded by the gene with GENE ID 852851 in the NCBI database. Aga2p, also known as the Aga2 protein, is a binding subunit of α-clusin that binds to the α-clusin core subunit, the Aga1 protein, via a disulfide bond. In some embodiments, Aga2p is a protein encoded by the gene with GENE ID 852851 in the NCBI database. In some embodiments, Aga2p has one, two, three, four, five, six, or more mutations in its amino acid sequence compared to the protein encoded by the gene with GENE ID 852851 in the NCBI database. In some embodiments, Aga2p does not have the S288C mutation in its amino acid sequence compared to the protein encoded by the gene with GENE ID 852851 in the NCBI database.In some embodiments, the amino acid sequence of Aga2p comprises the amino acid sequence set forth in SEQ ID NO: 1, or a conservatively substituted variant of the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of Aga2p is set forth in SEQ ID NO: 1.

[0151] In some embodiments, the single-chain domain is a peptide segment of 9 to 30 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) amino acids in length, and comprises at least one continuous register peptide of a fixed length of 9 amino acids that can be specifically recognized by the complex or a variant thereof that can be bound by the complex. In some embodiments, the amino acid sequence of the single-chain domain is as shown in any one of SEQ ID NOs: 2-6. In some embodiments, the amino acid sequence of the single-chain domain is a synonymous mutant of the amino acid sequence shown in any one of SEQ ID NOs: 2-6. In some embodiments, the amino acid sequence of the single-chain domain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 2-6. It should be understood that the introduction of mutations into the α chain and / or the β chain can make the registered peptide accommodated by the MHC II complex peptide binding domain constituted thereby longer or shorter, and the engineered cells comprising the α chain and the β chain introduced with the mutations should also be included in the scope of this application. Moreover, corresponding to the α chain and the β chain introduced with the mutations, the length of the registered peptide contained in the peptide chain can be more or less than 9 amino acids, for example, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

[0152] In some embodiments, the amino acid sequence of the registered peptide or a mutant thereof is shown in SEQ ID NO: 20-38.

[0153] In some embodiments, the first protein binding domain is non-covalently bound to the second protein binding domain. In some embodiments, the first protein binding domain is covalently bound to the second protein binding domain. The first protein binding domain and the second protein binding domain can be the same or different and can be homologous proteins or non-homologous proteins. In some embodiments, the first protein binding domain and the second protein binding domain comprise fragments from the same antibody. In some embodiments, the first protein binding domain and the second protein binding domain can be bound to each other by any method for forming antibody homologous or heterologous dimers (or polymers). The methods for forming antibody homo- or heterodimers (or multimers) include, but are not limited to, "knob-in-hole" technology (see, e.g., U.S. Patent Application No. 5,731,168), engineered electrostatic steering technology (see, e.g., PCT Application No. WO 2009 / 089004A1), leucine zipper technology (see, e.g., Kostelny et al, J. Immunol., 148(5): 1547-1553, 1992), and those described in U.S. Patent Application No. 4,676,980 and Brennan et al, Science, 229: 81, 1985. To achieve mutual binding based on the above methods, the first protein binding domain and the second protein binding domain may comprise mutations for the corresponding binding structures of the above methods. In some embodiments, the first protein binding domain and the second protein binding domain are bound to each other via a disulfide bond.

[0154] In some embodiments, the first protein-binding domain is a first leucine zipper domain, and the second protein-binding domain is a second leucine zipper domain, wherein the first leucine zipper domain and the second leucine zipper domain that form a complete leucine zipper can be used interchangeably in the first fusion protein and the second fusion protein; or the first protein-binding domain is an FcA domain, and the second protein-binding domain is an FcB domain, wherein the FcA domain and the FcB domain that form an FcAB dimer can be used interchangeably in the first fusion protein and the second fusion protein. That is, when the first protein-binding domain is a first leucine zipper domain, the second protein-binding domain is a second leucine zipper domain; and when the first protein-binding domain is a second leucine zipper domain, the second protein-binding domain is a first leucine zipper domain; or when the first protein-binding domain is FcA, the second protein-binding domain is FcB; and when the first protein-binding domain is FcB, the second protein-binding domain is FcA.

[0155] In some embodiments, the first leucine zipper domain and the second leucine zipper domain are both acidic leucine domains (Acid zipper). In some embodiments, the first leucine zipper domain and the second leucine zipper domain are both basic leucine domains (Basic zipper). In some embodiments, the first leucine zipper domain is an acidic leucine domain (Acid zipper), and the second leucine zipper domain is a basic leucine domain (Basic zipper). In some embodiments, the first leucine zipper domain and the second leucine zipper domain are both c-Jun leucine zipper domains (Jun Zipper). In some embodiments, the first leucine zipper domain is a Jun Zipper and the second leucine zipper domain is a c-Fos leucine zipper domain (Fos Zipper). In some embodiments, the acidic leucine domain comprises the amino acid sequence of SEQ ID NO: 7, or a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the amino acid sequence of the acidic leucine domain is the amino acid sequence of SEQ ID NO: 7, or a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0156] In some embodiments, the basic leucine domain comprises the amino acid sequence of SEQ ID NO: 8, or a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the amino acid sequence of the basic leucine domain is the amino acid sequence of SEQ ID NO: 8, or a conservatively substituted variant of the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0157] In some embodiments, the Fos Zipper comprises the amino acid sequence as set forth in SEQ ID NO: 9, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 9, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the amino acid sequence of the Fos Zipper is the amino acid sequence as set forth in SEQ ID NO: 9, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 9, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 9.

[0158] In some embodiments, the JunZipper comprises the amino acid sequence set forth in SEQ ID NO: 10, or a conservatively substituted variant of the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the amino acid sequence of the JunZipper is the amino acid sequence set forth in SEQ ID NO: 10, or a conservatively substituted variant of the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.

[0159] In some embodiments, the amino acid sequence of the first leucine zipper domain is shown in SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10;

[0160] Correspondingly, the amino acid sequence of the second leucine zipper domain is shown in SEQ ID NO: 8 or SEQ ID NO: 7 or SEQ ID NO: 10 or SEQ ID NO: 9.

[0161] In some embodiments, both the first and second protein-binding domains comprise the Fc domain of an immunoglobulin. Immunoglobulin molecules are split into Fc and Fab segments after papain digestion, with the Fc segment comprising the portion of the immunoglobulin molecule's heavy chain constant region excluding the CH1 segment. As used herein, the term "Fc domain" refers to a monomer and may refer to any portion of the constant region of any heavy chain of an immunoglobulin, excluding the CH1 segment. In some embodiments, the FcA and FcB may be derived from the Fc segment of the same immunoglobulin or from different immunoglobulins. In some embodiments, the immunoglobulins from which the FcA and FcB are derived may be selected from the group consisting of IgG1, IgG, IgE, IgM, IgD, and IgA. In some embodiments, the immunoglobulins from which the FcA and FcB are derived may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some embodiments, the FcA and FcB are selected from one or both of the amino acid sequences set forth in SEQ ID NOs: 11-19, conservatively substituted variants of the amino acid sequences set forth in SEQ ID NOs: 11-19, and amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 11-19. For example, in some embodiments, the amino acid sequences of the FcA and FcB are both set forth in SEQ ID NO: 11, or conservatively substituted variants of the amino acid sequences set forth in SEQ ID NO: 11, or amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 11. In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 12, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 12, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 12; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 13, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 13, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 13.In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO:4, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO:13, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:13; and the FcB has an amino acid sequence as set forth in SEQ ID NO:12, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO:12, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:3. In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 14, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 14, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 14; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 15, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 15, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15. In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 15, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 15, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 14, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 14, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 14.In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 16, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 16, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 16; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 17, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 17, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 17. In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 17, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 17, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 17; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 16, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 16, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 16. In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 18, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 18, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 18; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 19, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 19, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 19.In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 19, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 19, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 19; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 18, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 18, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 18.

[0162] In some embodiments, the FcA domain and FcB domain can increase the expression display amount and / or copy number of the complex. In some embodiments, the FcA domain only comprises a first CH3 domain, and the FcB domain only comprises a second CH3 domain; wherein the first CH3 domain and the second CH3 domain can be from the same immunoglobulin subtype or different immunoglobulin subtypes. In some embodiments, the FcA domain comprises both a first CH2 domain and a first CH3 domain, and the FcB domain comprises both a second CH2 domain and a second CH3 domain; wherein the first CH3 domain and the second CH3 domain may be derived from the same immunoglobulin subtype or different immunoglobulin subtypes; the first CH2 domain and the second CH2 domain may be derived from the same immunoglobulin subtype or different immunoglobulin subtypes; the first CH2 domain and the first CH3 domain may be derived from the same immunoglobulin subtype or different immunoglobulin subtypes, and the second CH2 domain and the second CH3 domain may be derived from the same immunoglobulin subtype or different immunoglobulin subtypes. Depending on the immunoglobulin source, for example, when the FcA and / or FcB are derived from IgE or IgM, in some embodiments, the FcA domain may further comprise a first CH4 domain, and the FcB domain may further comprise a second CH4 domain.

[0163] In some embodiments, the first CH2 domain and the first CH3 domain of the first protein-binding domain are directly or indirectly connected via a linker. In some embodiments, the second CH2 domain and the second CH3 domain of the first protein-binding domain are directly or indirectly connected via a linker. In some embodiments, the first CH2 domain and the first CH3 domain of the first protein-binding domain are indirectly connected via a linker; and the second CH2 domain and the second CH3 domain of the second protein-binding domain are indirectly connected via a linker. In some embodiments, the linker is a cleavable or non-cleavable linker. In some embodiments, the linker is a short peptide or a connecting peptide, which can be a flexible connecting peptide or a rigid connecting peptide. Those skilled in the art can select a connecting peptide according to their needs. The connecting peptide can be selected from different lengths or functions. The types, functions and characteristics of the connecting peptide can be referenced, for example, Reddy Chichili VP, Kumar V, Sivaraman J. Linkers in the structural biology of protein-protein interactions. Protein Sci. 2013; 22(2): 153-167. doi: 10.1002 / pro.2206.

[0164] In some embodiments, either or both of the FcA domain and the FcB domain comprise one or more amino acid modifications that (1) enhance or stabilize the FcA domain and / or the FcB domain itself ("FcS"), and / or (2) enhance or stabilize the covalent or non-covalent binding between the FcA domain and the FcB domain ("FcM"), and / or (3) reduce or avoid various possible mispairing of the α chain and β chain of claim 1 resulting from mispairing of the FcA domain and the FcB domain ("FcN").

[0165] In some embodiments, the first protein binding domain is linked to the C-terminus of the α chain, and the second protein binding domain is linked to the C-terminus of the β chain.

[0166] Preferably, the signal peptide for initiating expression of the first fusion protein and the second fusion protein is SPP SP or AGA2 SP. More preferably, the amino acid sequence of AGA2 SP is shown in SEQ ID NO: 39, and the amino acid sequence of SPP SP is shown in SEQ ID NO: 45.

[0167] In some embodiments, the complex is an MHC class I molecule or an MHC class II molecule. In some embodiments, the complex is an HLA class I molecule or an HLA class II molecule.

[0168] In some embodiments, the α chain is encoded by HLA-DRA*01 or other alleles of the HLA-DRA family, and the β chain is encoded by HLA-DRB1*01 or HLA-DRB1*03 or HLA-DRB1*04 or HLA-DRB1*15 or other alleles of the HLA-DRB family; or

[0169] The α chain is encoded by HLA-DQA1*01 or HLA-DQA1*03 or HLA-DQA1*05 or other alleles of the HLA-DQA1 family, and the β chain is encoded by HLA-DQB1*02 or HLA-DQB1*03 or HLA-DQB1*05 or HLA-DQB1*06 or other alleles of the HLA-DQB1 family; or

[0170] The α chain is encoded by HLA-DPA1*01:03 or HLA-DPA1*02:02 or other alleles of the HLA-DPA1 family, and the β chain is encoded by HLA-DPB1*01:01 or HLA-DPB1*02:01 or HLA-DPB1*04:01 or HLA-DPB1*04:02 or other alleles of the HLA-DPB1 family.

[0171] Engineered nucleic acid molecules

[0172] In the second aspect of the present application, there is provided an engineered nucleic acid molecule encoding a major histocompatibility complex (MHC), wherein the complex comprises an α chain and a β chain, wherein the α chain is connected to a first protein binding domain, and the β chain is connected to a second protein binding domain, wherein the α chain and the β chain are bound to a single-chain domain by a non-covalent bond, and the first protein binding domain is bound to the second protein binding domain to enhance or promote the formation of a trimer composed of the complex and the single-chain domain. At the same time, the present application also provides a nucleic acid molecule encoding the trimer. The trimer is a trimer composed of the MHC and the single-chain domain contained in the engineered cell described in the first aspect of the present application. The MHC is the MHC contained in the engineered cell described in the first aspect of the present application.

[0173] In some embodiments, the engineered nucleic acid molecule is an engineered DNA molecule. In some embodiments, the DNA molecule can be replicated and / or expressed in a cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a eukaryotic cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a prokaryotic cell. In some embodiments, the DNA molecule can be expressed in a eukaryotic cell and can be replicated in a prokaryotic cell. Thus, in addition to comprising a nucleic acid fragment encoding the MHC or trimer, the DNA molecule also comprises genetic manipulation or regulatory elements for replication and / or expression in prokaryotic and / or eukaryotic cells.

[0174] The engineered DNA molecule is made to replicate or efficiently replicate the necessary structural elements in the cell, which are known in the art and include, for example, an origin of replication (ORI). In some embodiments, the engineered DNA molecule further includes a marker gene or its fragment and / or a reporter gene or its fragment and a unique restriction endonuclease site that allows insertion of the DNA element, preferably a restriction endonuclease site in the form of a multiple cloning site (MCS). The marker gene is conducive to identifying cells containing the plasmid that comprises the marker gene and can be selected from, for example, antibiotic resistance genes. Each restriction endonuclease site in the MCS can be specifically recognized by different restriction endonucleases.

[0175] In some embodiments, the DNA molecule is a DNA plasmid. As used herein, the term "DNA plasmid" refers to a plasmid consisting of a double-stranded DNA molecule. In some embodiments, the "plasmid" is a circular DNA molecule. In some embodiments, the "plasmid" can also encompass linear DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by, for example, cutting a circular plasmid with a restriction endonuclease, thereby converting the circular plasmid molecule into a linear molecule and linearizing the circular plasmid, as well as linear molecules that can be replicated in prokaryotes. Plasmids can replicate, i.e., amplify the genomic genetic information stored in the nucleoid or nucleoid of a prokaryotic cell in a cell, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. Preferably, the DNA plasmid according to the present invention is a medium copy or high copy plasmid, more preferably a high copy plasmid. Examples of such high copy plasmids are vectors based on pUC, pTZ plasmids, or any other plasmid (e.g., pMB1, pCoIE1) containing an ORI that supports high copies of the plasmid.

[0176] In some embodiments, the engineered DNA molecule is a DNA molecule or a fragment thereof constituting a nucleoid or nucleoid of a prokaryotic organism, or a DNA molecule or a fragment thereof constituting a eukaryotic genome, that is, the coding sequence comprising the aforementioned MHC or trimer or its complementary sequence can be replicated along with the prokaryotic genome.

[0177] In some embodiments, the engineered DNA molecule can be transcribed into mRNA. In some embodiments, the engineered DNA molecule further comprises coding sequences for elements that can be used to initiate or regulate expression of the protein, polypeptide, or fragment thereof after transcription, including but not limited to a 5'UTR, a 3'UTR, a poly(A) tail (or a poly(A) tail signal), etc. In some embodiments, the engineered DNA molecule comprises a coding sequence for at least one untranslated region (UTR). In some embodiments, the engineered DNA molecule comprises at least a coding sequence for a 5'UTR and a coding sequence for the protein, polypeptide, or fragment thereof. In some embodiments, the engineered DNA molecule comprises, from 5' to 3', at least the coding sequence for the 5'UTR, the coding sequence for the MHC or trimer, the coding sequence for the 3'UTR, a poly(A) tail signal (or a DNA sequence corresponding to a poly(A) tail sequence), and may further comprise a start codon (5' end) and a stop codon (3' end) at either end of the coding sequence for the MHC or trimer, which are the first three nucleotides and last three nucleotides of the mRNA molecule that can be translated, respectively. 5'UTR generally comprises at least one ribosome binding site (RBS), such as the Shine-Dalgarno sequence in prokaryotes, or at least one translation initiation site, such as the Kozak sequence in eukaryotes. RBS promotes the efficient and accurate translation of mRNA molecules by recruiting ribosomes at the time of translation initiation. Its activity can be optimized by changing the length and sequence of a given RBS or translation revelation site and the distance from the start codon. Alternatively or optionally, 5'UTR includes an internal ribosome entry site or IRES. 3'UTR may comprise one or more regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of the mRNA molecule, binding sites for regulatory RNA molecules (such as miRNA molecules), and / or signal sequences that participate in the intracellular transport of mRNA molecules.

[0178] On the basis of the aforementioned embodiment, in some embodiments, the target gene fragment further comprises one or more additional regulatory sequences, such as binding sites for amino acid sequences that enhance mRNA molecule stability, binding sites for amino acid sequences that enhance mRNA molecule translation, regulatory elements (such as riboswitches), and / or nucleotide sequences that have a positive impact on translation initiation. In addition, within the 5'UTR, preferably there is no functional upstream open reading frame, out-of-frame upstream translation initiation site, out-of-frame upstream start codon, and / or nucleotide sequences that produce secondary structures that reduce or prevent translation. The presence of such nucleotide sequences in the 5'UTR can have a negative impact on translation.

[0179] The coding sequence of the MHC or trimer comprises codons that can be translated into an amino acid sequence. The coding sequence may comprise all naturally occurring codons encoding amino acids, or may comprise some or all artificially synthesized codons. In some embodiments, some or all of the codons are codon-optimized. In some embodiments, some or all of the codons encode unnatural amino acids.

[0180] In some embodiments, the engineered DNA molecule further comprises structural elements on the 5' end of the target gene segment that are necessary to initiate or regulate transcription of the RNA, and these structural elements are known in the art. In some embodiments, the structural elements comprise at least a promoter. Promoters and their sequences are known in the art and include weak promoters, medium-strength promoters, strong promoters, mini promoters, and core promoters. In certain specific embodiments, the promoter is a strong promoter. In some embodiments, the promoter can initiate transcription of the coding sequence for the MHC or trimer in prokaryotes. In some embodiments, the promoter can initiate transcription of the coding sequence for the MHC or trimer in eukaryotic cells. The "promoter" comprises at least one transcriptional recognition site followed by a transcription factor binding site. The recognition and binding sites can interact with amino acid sequences that mediate or regulate transcription. The binding site is located closer to the target gene segment than the recognition site. The binding site can be, for example, a Pribnow box in prokaryotes or a TATA box in eukaryotes. For example, in some embodiments, when using the Pribnow box, the transcription recognition site can be located at about 35bp upstream of the transcription start site, and the transcription factor binding site can be located at about 10bp upstream of the transcription start site. In some embodiments, the promoter comprises at least one other regulatory element, such as an upstream element rich in AT at about 40 and / or 60 nucleotides before the transcription start site, and / or an additional regulatory element that enhances promoter activity between the recognition site and the binding site. In some embodiments, the promoter is a strong promoter, that is, the promoter comprises a sequence that promotes transcription of the aforementioned RNA coding sequence. Strong promoters are well known to those skilled in the art, such as OXB18, OXB19 and OXB20 promoters derived from the RecA promoter of Escherichia coli, or can be identified or synthesized by conventional laboratory procedures. In some embodiments, the promoter is a T7 promoter. In some embodiments, the promoter also comprises additional regulatory elements before it, such as an enhancer that can promote transcription of the aforementioned RNA coding sequence in a DNA plasmid.

[0181] In some embodiments, the eukaryotic cell is a yeast cell.In some embodiments, the DNA molecule is a yeast display vector.

[0182] In addition, the present application also provides an RNA molecule encoding the above-mentioned MHC. In some embodiments, the RNA molecule is transcribed from the above-mentioned engineered DNA molecule.

[0183] In some embodiments, the engineered nucleic acid molecule can also be a hybrid molecule of DNA and RNA.

[0184] In addition, the present application also covers any cells comprising the above-mentioned engineered nucleic acid molecules.

[0185] Preparation and identification methods

[0186] The present application also relates to a method for preparing a major histocompatibility complex (MHC) or a trimer formed by non-covalent association of an MHC and a single-chain domain, the method comprising transforming a cell with the engineered nucleic acid molecule of the second aspect, and culturing the cell under conditions that express the complex or trimer. In some embodiments, the cell is a yeast cell, such as a Saccharomyces cerevisiae cell. In some embodiments, the cell is a yeast cell and the engineered nucleic acid molecule is a yeast display vector. In some embodiments, the cell is a yeast strain EBY100.

[0187] The present application also relates to a method for identifying a peptide that binds to a major histocompatibility complex (MHC), the method comprising:

[0188] i) causing the cell of the first aspect to display the single-chain domain library, and

[0189] ii) detecting trimers on the surface of monoclonal cells in the cell library displaying the single-chain domain, thereby identifying peptides that bind to the complex. In some embodiments, the single-chain domain comprises a peptide, or a mutant of a peptide, or a library of mutants of a peptide, or a mixture of peptides. In some embodiments, the peptide, or a mutant of a peptide, or a library of mutants of a peptide, or a mixture of peptides non-covalently binds to the complex. In some embodiments, the peptide is a peptide segment of 9 to 30 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) amino acids in length, and comprises at least one continuous registered peptide of a fixed length of 9 amino acids that can be specifically recognized by the complex or a variant thereof that can be bound by the complex.

[0190] The inventors expressed human MHC alleles as native, non-covalent α / β dimers on the surface of yeast cells to enable rapid flow cytometry-based screening of peptide ligands directly from selected antigens and to improve the sensitivity and accuracy of peptide / MHC affinity assays.

[0191] This application constructs the RIPAAH (Rapid Identification of Peptide Antigen Associated by HLA-II) method and related cells and nucleic acids, that is, using a yeast display platform to eliminate labor-intensive expression / purification steps. As a unicellular eukaryote, yeast cells have the fast and easy cloning characteristics of Escherichia coli and are equipped with a post-translational modification mechanism similar to that of mammalian or insect cells. This method connects one chain (α or β) of a given MHC-II allele to a yeast surface protein and allows the other chain (α or β) to be secreted by the same yeast cell as a soluble component. In this scheme, the two C-termini of the α / β chain can be modified to carry the amino acid sequence (FcAB) of the antibody heavy chain crystallizable fragment, which promotes the pairing of the soluble secretory chain with other surface anchor chains. The unique design of this method for the surface expression of non-covalent MHC-IIα / β heterodimers does not require covalently linked positioning peptides, interchain linkers or MHC-II amino acid residue mutations. This method successfully displays correctly folded and fully functional MHC-II proteins on the surface of yeast cells. This method not only effectively improves the sensitivity and accuracy of determining the affinity between the target peptide and the MHC-II protein displayed by yeast, but also relies on the competitive binding between the test peptide and the reference peptide and the MHC-II protein displayed by yeast to achieve short-term and efficient high-throughput determination.

[0192] With this method, cells, and nucleic acids, the inventors can more efficiently create yeast cell clone libraries, allowing different human MHC-II alleles to be expressed in their native form on the surface of different yeast clones for TCR epitope peptide screening. The emergence of this method will provide an effective solution for the rapid development of new vaccines and cell therapies based on T cell immunity. In addition, high-throughput and high-content affinity determination of MHC-II / peptide ligands on the surface of single-cell yeast will advance artificial intelligence computational methods for T cell epitope peptide map discovery.

[0193] cell

[0194] In one aspect, the present application provides a cell comprising a major histocompatibility complex (MHC), wherein the complex comprises an α chain and a β chain, wherein:

[0195] The α chain is connected to the first protein binding domain to form a first fusion protein,

[0196] The β chain is connected to a second protein binding domain to form a second fusion protein,

[0197] The first fusion protein binds to the second fusion protein to form the complex,

[0198] The first protein binding domain and the second protein binding domain (eg, forming an FcAB dimer) enhance the binding of the α chain and the β chain to form the MHC.

[0199] The cells are engineered cells.

[0200] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a cell derived from a unicellular organism. In some embodiments, the cell is derived from a unicellular eukaryotic organism. In some embodiments, the cell is a yeast cell. In some embodiments, the yeast is Saccharomyces cerevisiae. In some embodiments, the yeast is strain EBY100 (Boder & Wittrup, 1997).

[0201] In some embodiments, the complex is bound to the surface of the cell by linking the α chain or the β chain to a molecule on the cell surface. In some embodiments, the additional α chain or the additional β chain in the complex is bound to the molecule on the cell surface by non-covalently binding to the β chain or the α chain. That is, the α chain and the β chain synthesized by different cells can bind to each other. The α chain synthesized by one cell can bind to the β chain synthesized by another cell; the β chain synthesized by the cell can also bind to the α chain synthesized by another cell, thereby allowing the cell membrane to contain a complete MHC. In order to achieve a combination of α chains and β chains secreted by different cells, in some embodiments, at least one of the α chain and the β chain, or more precisely, the first fusion protein or the second fusion protein, is secretable.

[0202] The molecule can be any molecule that can bind the first protein binding domain and / or the second protein binding domain to the cell surface. In some embodiments, the molecule and the first protein binding domain and / or the second protein binding domain are bound to each other by a covalent bond or a non-covalent bond. In some embodiments, the molecule is a protein. In some embodiments, the molecule is a protein, and the first protein binding domain and / or the second protein binding domain are bound to the molecule by a peptide bond. In some embodiments, the molecule comprises a structure that can anchor the yeast cell wall. In some embodiments, the molecule comprises a structure that can anchor the yeast cell wall and a secretion signal peptide (or secretion signal region). In some embodiments, the structure that can anchor the yeast cell wall can directly or indirectly bind to cell wall glucan or cell wall mannan. In some embodiments, the structure that can anchor the yeast cell wall can directly or indirectly bind to cell wall glucan or cell wall mannan covalently or non-covalently. In some embodiments, the structure that can anchor the yeast cell wall can directly or indirectly bind to cell wall glucan covalently or bind to cell wall mannan non-covalently. In some embodiments, the yeast cell wall-anchorable structure and / or secretion signal peptide is derived from the α-lectin system and / or the floccin system (glycosylphosphatidylinositol anchor system and floccin domain anchor system). In some embodiments, the yeast cell wall-anchorable structure is directly or indirectly associated with or linked to a GPI anchor attachment signal region or a flocculation functional region. In some embodiments, the molecule is endogenous to the cell. In some embodiments, the molecule is selected from the group consisting of: Aga2p, α-lectin, α-lectin, floccin, Cwp1p, Cwp2p, or Tip1p. In some embodiments, the protein is Aga2p. In some embodiments, Aga2p is a protein encoded by the gene with GENE ID 852851 in the NCBI database. Aga2p, also known as the Aga2 protein, is a binding subunit of α-lectin that can bind to the α-lectin core subunit, the Aga1 protein, via a disulfide bond. In some embodiments, Aga2p is a protein encoded by the gene with GENE ID 852851 in the NCBI database. In some embodiments, Aga2p has one, two, three, four, five, six or more mutations in its amino acid sequence compared to the protein encoded by the gene with GENE ID 852851 in the NCBI database. In some embodiments, Aga2p does not have the S288C mutation in its amino acid sequence compared to the protein encoded by the gene with GENE ID 852851 in the NCBI database.In some embodiments, the amino acid sequence of Aga2p comprises the amino acid sequence set forth in SEQ ID NO: 1, or a conservatively substituted variant of the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of Aga2p is set forth in SEQ ID NO: 1.

[0203] In some embodiments, the first protein binding domain is non-covalently bound to the second protein binding domain. In some embodiments, the first protein binding domain is covalently bound to the second protein binding domain. The first protein binding domain and the second protein binding domain can be the same or different and can be homologous proteins or non-homologous proteins. In some embodiments, the first protein binding domain and the second protein binding domain comprise fragments from the same antibody. In some embodiments, the first protein binding domain and the second protein binding domain can be bound to each other by any method for forming antibody homologous or heterologous dimers (or polymers). The methods for forming antibody homo- or heterodimers (or multimers) include, but are not limited to, "knob-in-hole" technology (see, e.g., U.S. Patent Application No. 5,731,168), engineered electrostatic steering technology (see, e.g., PCT Application No. WO 2009 / 089004A1), leucine zipper technology (see, e.g., Kostelny et al, J. Immunol., 148(5):1547-1553, 1992), and those described in U.S. Patent Application No. 4,676,980 and Brennan et al, Science, 229:81, 1985. To achieve mutual binding based on the above methods, the first protein binding domain and the second protein binding domain may comprise mutations for the corresponding binding structures of the above methods. In some embodiments, the first protein binding domain and the second protein binding domain are bound to each other via a disulfide bond.

[0204] In some embodiments, the first protein binding domain and the second protein binding domain both comprise the Fc domain of an immunoglobulin. After papain digestion, an immunoglobulin molecule is split into an Fc segment and a Fab segment, wherein the Fc segment includes the portion other than the CH1 segment of the heavy chain constant region of the immunoglobulin molecule. In this application, the term "Fc domain" is a monomer that can refer to any portion of the constant region of any heavy chain of an immunoglobulin other than the CH1 segment. In this application, when the first protein binding domain and the second protein binding domain both comprise the Fc domain of an immunoglobulin, the Fc domain in the first protein binding domain is referred to as FcA, and the Fc domain in the second protein binding domain is referred to as FcB. The FcA domain and the FcB domain may have the same or different amino acid sequences, and the FcA domain and the FcB domain that form the FcAB dimer may be used interchangeably in the first fusion protein and the second fusion protein. That is, when the first protein-binding domain is FcA, the second protein-binding domain is FcB; and when the first protein-binding domain is FcB, the second protein-binding domain is FcA. In some embodiments, the FcA and FcB can be derived from the Fc segment of the same immunoglobulin, or from the Fc segments of different immunoglobulins. In some embodiments, the immunoglobulins from which the FcA and FcB are derived can be selected from the group consisting of IgG1, IgG, IgE, IgM, IgD, and IgA. In some embodiments, the immunoglobulins from which the FcA and FcB are derived can be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some embodiments, the FcA and FcB are selected from one or both of the amino acid sequences set forth in SEQ ID NOs: 11-19, conservatively substituted variants of the amino acid sequences set forth in SEQ ID NOs: 11-19, and amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 11-19. For example, in some embodiments, the amino acid sequences of the FcA and FcB are both set forth in SEQ ID NO: 11, or conservatively substituted variants of the amino acid sequences set forth in SEQ ID NO: 11, or amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 11.In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 12, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 12, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 12; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 13, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 13, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 13, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 13, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 13; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 12, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 12, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 14, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 14, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 14; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 15, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 15, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15.In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 15, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 15, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 15; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 14, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 14, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 14. In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 16, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 16, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 16; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 17, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 17, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 17. In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 17, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 17, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 17; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 16, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 16, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 16.In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 18, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 18, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 18; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 19, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 19, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 19. In some embodiments, the FcA has an amino acid sequence as set forth in SEQ ID NO: 19, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 19, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 19; and the FcB has an amino acid sequence as set forth in SEQ ID NO: 18, or a conservatively substituted variant of the amino acid sequence as set forth in SEQ ID NO: 18, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 18.

[0205] In some embodiments, the FcA domain and FcB domain can increase the expression display amount and / or copy number of the complex. In some embodiments, the FcA domain only comprises a first CH3 domain, and the FcB domain only comprises a second CH3 domain; wherein the first CH3 domain and the second CH3 domain can be from the same immunoglobulin subtype or different immunoglobulin subtypes. In some embodiments, the FcA domain comprises both a first CH2 domain and a first CH3 domain, and the FcB domain comprises both a second CH2 domain and a second CH3 domain; wherein the first CH3 domain and the second CH3 domain may be derived from the same immunoglobulin subtype or different immunoglobulin subtypes; the first CH2 domain and the second CH2 domain may be derived from the same immunoglobulin subtype or different immunoglobulin subtypes; the first CH2 domain and the first CH3 domain may be derived from the same immunoglobulin subtype or different immunoglobulin subtypes, and the second CH2 domain and the second CH3 domain may be derived from the same immunoglobulin subtype or different immunoglobulin subtypes. Depending on the immunoglobulin source, for example, when the FcA and / or FcB are derived from IgE or IgM, in some embodiments, the FcA domain may further comprise a first CH4 domain, and the FcB domain may further comprise a second CH4 domain.

[0206] In some embodiments, the first CH2 domain and the first CH3 domain of the first protein-binding domain are directly or indirectly connected via a linker. In some embodiments, the second CH2 domain and the second CH3 domain of the first protein-binding domain are directly or indirectly connected via a linker. In some embodiments, the first CH2 domain and the first CH3 domain of the first protein-binding domain are indirectly connected via a linker; and the second CH2 domain and the second CH3 domain of the second protein-binding domain are indirectly connected via a linker. In some embodiments, the linker is a cleavable or non-cleavable linker. In some embodiments, the linker is a short peptide or a connecting peptide, which can be a flexible connecting peptide or a rigid connecting peptide. Those skilled in the art can select a connecting peptide according to their needs. The connecting peptide can be selected from different lengths or functions. The types, functions and characteristics of the connecting peptide can be referenced, for example, Reddy Chichili VP, Kumar V, Sivaraman J. Linkers in the structural biology of protein-protein interactions. Protein Sci. 2013; 22(2): 153-167. doi: 10.1002 / pro.2206.

[0207] In some embodiments, either or both of the FcA domain and the FcB domain comprise one or more amino acid modifications that enhance or stabilize the FcA domain and / or the FcB domain itself ("FcS"), and / or enhance or stabilize the covalent or non-covalent binding between the FcA domain and the FcB domain ("FcM"), and / or reduce or avoid the various possible mispairings of the α chain and β chain mentioned herein resulting from mispairing of the FcA domain and the FcB domain ("FcN").

[0208] In some embodiments, the first protein binding domain is linked to the C-terminus of the α chain, and the second protein binding domain is linked to the C-terminus of the β chain.

[0209] Preferably, the signal peptide for initiating expression of the first fusion protein and the second fusion protein is SPP SP or AGA2 SP. More preferably, the amino acid sequence of AGA2 SP is shown in SEQ ID NO: 39, and the amino acid sequence of SPP SP is shown in SEQ ID NO: 45.

[0210] In some embodiments, the complex is an MHC class I molecule or an MHC class II molecule. In some embodiments, the complex is an HLA class I molecule or an HLA class II molecule.

[0211] In some embodiments, the α chain is encoded by HLA-DRA*01 or other alleles of the HLA-DRA family, and the β chain is encoded by HLA-DRB1*01 or HLA-DRB1*03 or HLA-DRB1*04 or HLA-DRB1*15 or other alleles of the HLA-DRB family; or

[0212] The α chain is encoded by HLA-DQA1*01 or HLA-DQA1*03 or HLA-DQA1*05 or other alleles of the HLA-DQA1 family, and the β chain is encoded by HLA-DQB1*02 or HLA-DQB1*03 or HLA-DQB1*05 or HLA-DQB1*06 or other alleles of the HLA-DQB1 family; or

[0213] The α chain is encoded by HLA-DPA1*01:03 or HLA-DPA1*02:02 or other alleles of the HLA-DPA1 family, and the β chain is encoded by HLA-DPB1*01:01 or HLA-DPB1*02:01 or HLA-DPB1*04:01 or HLA-DPB1*04:02 or other alleles of the HLA-DPB1 family.

[0214] Engineered nucleic acid molecules

[0215] In a second aspect of the present application, an engineered nucleic acid molecule encoding a major histocompatibility complex (MHC) is provided, wherein the nucleic acid encoding the major histocompatibility complex (MHC) is the histocompatibility complex contained in the cell of the first aspect of the present application. The engineered nucleic acid molecule encoding the MHC refers to an engineered nucleic acid molecule comprising the MHC coding sequence and / or its complementary sequence.

[0216] Specifically, in some embodiments, the complex comprises an α chain and a β chain, wherein the α chain is connected to a first protein binding domain, and the β chain is connected to a second protein binding domain, wherein the first protein binding domain and the second protein binding domain are capable of binding to form the complex, and the first protein binding domain and the second protein binding domain form an FcAB dimer to enhance the binding of the α chain and the β chain to form the MHC.

[0217] In some embodiments, the engineered nucleic acid molecule is an engineered DNA molecule. In some embodiments, the DNA molecule can be replicated and / or expressed in a cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a eukaryotic cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a prokaryotic cell. In some embodiments, the DNA molecule can be expressed in a eukaryotic cell and can be replicated in a prokaryotic cell. Therefore, in addition to comprising a nucleic acid fragment encoding the major histocompatibility complex (MHC), the DNA molecule also comprises genetic manipulations or regulatory elements for replication and / or expression in prokaryotic and / or eukaryotic cells.

[0218] The engineered DNA molecule is made to replicate or efficiently replicate the necessary structural elements in the cell, which are known in the art and include, for example, an origin of replication (ORI). In some embodiments, the engineered DNA molecule further includes a marker gene or its fragment and / or a reporter gene or its fragment and a unique restriction endonuclease site that allows insertion of the DNA element, preferably a restriction endonuclease site in the form of a multiple cloning site (MCS). The marker gene is conducive to identifying cells containing the plasmid that comprises the marker gene and can be selected from, for example, antibiotic resistance genes. Each restriction endonuclease site in the MCS can be specifically recognized by different restriction endonucleases.

[0219] In some embodiments, the DNA molecule is a DNA plasmid. As used herein, the term "DNA plasmid" refers to a plasmid consisting of a double-stranded DNA molecule. In some embodiments, the "plasmid" is a circular DNA molecule. In some embodiments, the "plasmid" can also encompass linear DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by, for example, cutting a circular plasmid with a restriction endonuclease, thereby converting the circular plasmid molecule into a linear molecule and linearizing the circular plasmid, as well as linear molecules that can be replicated in prokaryotes. Plasmids can replicate, i.e., amplify the genomic genetic information stored in the nucleoid or nucleoid of a prokaryotic cell in a cell, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. Preferably, the DNA plasmid according to the present invention is a medium copy or high copy plasmid, more preferably a high copy plasmid. Examples of such high copy plasmids are vectors based on pUC, pTZ plasmids, or any other plasmid (e.g., pMB1, pCoIE1) containing an ORI that supports high copies of the plasmid.

[0220] In some embodiments, the engineered DNA molecule is a DNA molecule or a fragment thereof constituting a nucleoid or nucleoid of a prokaryotic organism, or a DNA molecule or a fragment thereof constituting a eukaryotic genome, that is, the coding sequence comprising the aforementioned MHC or its complementary sequence can be replicated along with the prokaryotic genome.

[0221] In some embodiments, the engineered DNA molecule can be transcribed into mRNA. In some embodiments, the engineered DNA molecule further comprises coding sequences for elements that can be used to initiate or regulate expression of the protein, polypeptide, or fragment thereof after transcription, including but not limited to a 5'UTR, a 3'UTR, a poly(A) tail (or a poly(A) tail signal), etc. In some embodiments, the engineered DNA molecule comprises coding sequences for at least one untranslated region (UTR). In some embodiments, the engineered DNA molecule comprises at least the coding sequence for the 5'UTR and the coding sequence for the protein, polypeptide, or fragment thereof. In some embodiments, the engineered DNA molecule comprises, in order from 5' to 3', at least the coding sequence for the 5'UTR, the coding sequence for the MHC, the coding sequence for the 3'UTR, and a poly(A) tail signal (or a DNA sequence corresponding to the poly(A) tail sequence), and may further comprise a start codon (5' end) and a stop codon (3' end) at either end of the MHC coding sequence, which are the first three nucleotides and last three nucleotides of the mRNA molecule that can be translated, respectively. 5'UTR generally comprises at least one ribosome binding site (RBS), such as the Shine-Dalgarno sequence in prokaryotes, or at least one translation initiation site, such as the Kozak sequence in eukaryotes. RBS promotes the efficient and accurate translation of mRNA molecules by recruiting ribosomes at the time of translation initiation. Its activity can be optimized by changing the length and sequence of a given RBS or translation revelation site and the distance from the start codon. Alternatively or optionally, 5'UTR includes an internal ribosome entry site or IRES. 3'UTR may comprise one or more regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of the mRNA molecule, binding sites for regulatory RNA molecules (such as miRNA molecules), and / or signal sequences that participate in the intracellular transport of mRNA molecules.

[0222] On the basis of the aforementioned embodiment, in some embodiments, the target gene fragment further comprises one or more additional regulatory sequences, such as binding sites for amino acid sequences that enhance mRNA molecule stability, binding sites for amino acid sequences that enhance mRNA molecule translation, regulatory elements (such as riboswitches), and / or nucleotide sequences that have a positive impact on translation initiation. In addition, within the 5'UTR, preferably there is no functional upstream open reading frame, out-of-frame upstream translation initiation site, out-of-frame upstream start codon, and / or nucleotide sequences that produce secondary structures that reduce or prevent translation. The presence of such nucleotide sequences in the 5'UTR can have a negative impact on translation.

[0223] The MHC coding sequence comprises codons that can be translated into an amino acid sequence. The coding sequence may comprise all naturally occurring codons encoding amino acids, or some or all of the codons may be composed of synthetic codons. In some embodiments, some or all of the codons are codon-optimized. In some embodiments, some or all of the codons encode unnatural amino acids.

[0224] In some embodiments, the engineered DNA molecule further comprises structural elements on the 5' end of the target gene segment that are necessary to initiate or regulate transcription of the RNA, and these structural elements are known in the art. In some embodiments, the structural elements comprise at least a promoter. Promoters and their sequences are known in the art and include weak promoters, medium-strength promoters, strong promoters, mini promoters, and core promoters. In certain specific embodiments, the promoter is a strong promoter. In some embodiments, the promoter can initiate transcription of the MHC coding sequence in prokaryotes. In some embodiments, the promoter can initiate transcription of the MHC coding sequence in eukaryotes. The "promoter" comprises at least one transcription recognition site followed by a transcription factor binding site. The recognition and binding sites can interact with amino acid sequences that mediate or regulate transcription. The binding site is located closer to the target gene segment than the recognition site. The binding site can be, for example, a Pribnow box in prokaryotes or a TATA box in eukaryotes. For example, in some embodiments, when using the Pribnow box, the transcription recognition site can be located at about 35bp upstream of the transcription start site, and the transcription factor binding site can be located at about 10bp upstream of the transcription start site. In some embodiments, the promoter comprises at least one other regulatory element, such as an upstream element rich in AT at about 40 and / or 60 nucleotides before the transcription start site, and / or an additional regulatory element that enhances promoter activity between the recognition site and the binding site. In some embodiments, the promoter is a strong promoter, that is, the promoter comprises a sequence that promotes transcription of the aforementioned RNA coding sequence. Strong promoters are well known to those skilled in the art, such as OXB18, OXB19 and OXB20 promoters derived from the RecA promoter of Escherichia coli, or can be identified or synthesized by conventional laboratory procedures. In some embodiments, the promoter is a T7 promoter. In some embodiments, the promoter also comprises additional regulatory elements before it, such as an enhancer that can promote transcription of the aforementioned RNA coding sequence in a DNA plasmid.

[0225] In some embodiments, the eukaryotic cell is a yeast cell.In some embodiments, the DNA molecule is a yeast display vector.

[0226] In addition, the present application also provides an RNA molecule encoding the above-mentioned MHC. In some embodiments, the RNA molecule is transcribed from the above-mentioned engineered DNA molecule.

[0227] In some embodiments, the engineered nucleic acid molecule can also be a hybrid molecule of DNA and RNA.

[0228] In addition, the present application also covers any cells comprising the above-mentioned engineered nucleic acid molecules.

[0229] Preparation and identification methods

[0230] The present application also relates to a method for preparing a major histocompatibility complex (MHC), comprising: transforming a cell with the engineered nucleic acid molecule of the second aspect described above, and culturing the cell under conditions that express the complex. In some embodiments, the cell is a yeast cell, such as a Saccharomyces cerevisiae cell. In some embodiments, the cell is a yeast cell and the engineered nucleic acid molecule is a yeast display vector.

[0231] The present application also relates to a method for identifying a peptide that binds to a major histocompatibility complex (MHC), the method comprising:

[0232] i) contacting the cell according to the first aspect with a peptide,

[0233] and ii) detecting binding of the peptide to the complex, thereby identifying the peptide bound to the complex.

[0234] In some embodiments, the peptide comprises a mixture of peptides.

[0235] In some embodiments, the peptide competes with a reference peptide for binding to the complex. The "reference peptide" can be any peptide known to bind to MHC, such as an antigen epitope peptide.

[0236] Example

[0237] Example 1: Yeast Cell Co-Display Relies on Peptide Binding and HLA-Linked C-Terminal Domain Enabling

[0238] As a transmembrane protein, the stability of MHC-II heterodimerization of its α and β chains depends to a certain extent on the cooperation of their respective transmembrane domains and intracellular domains. In order to use eukaryotic single-cell organisms such as yeast to express stable human MHC-II extracellular domains (excluding the transmembrane domain and intracellular domain), the inventors first selected protein heterodimers corresponding to the most representative alleles of MHC-II human leukocyte antigens, such as HLA-DR1 (HLA-DRA*01:01 / HLA-DRB1*01:01, as shown in SEQ ID NO:40 and SEQ ID NO:41), HLA-DR4 (HLA-DRA*01:01 / HLA-DRB1*04:01, as shown in SEQ ID NO:40 and SEQ ID NO:42), and HLA-DQ6 (HLA-DQA1*01:02 / HLA-DQB1*06:02, as shown in SEQ ID NO:43 and SEQ ID NO:44), and then constructed yeast shuttle vectors that can express DR1, DR4, or DQ6 recombinant extracellular domain complexes. The bidirectional GAL1-10 promoter in the designed vector plasmid directs simultaneous expression of both the α and β chains. Furthermore, a leucine zipper domain (LZA or LZB to form a stable LZ dimer, as shown in SEQ ID NOs: 9 and 10 in this example) or an antibody heavy chain constant domain (FcA or FcB to form a stable Fc dimer, as shown in SEQ ID NOs: 15 and 16 in this example) is fused to the C-terminus of each of the expressed α and β chains. This facilitates the formation of native HLA complexes with HLA-II α / β chains bearing LZ or Fc modifications (Figures 2-7). Furthermore, the synthetic SPP SP (as shown in SEQ ID NO: 45) signal peptide is replaced with the endogenous yeast AGA2 SP (as shown in SEQ ID NO: 39) signal peptide at the N-terminus of the Fc-modified DR β chain or DQ α chain. Although both C-terminally fused LZ and Fc can promote stable heterodimerization of HLA α and β chains, flow cytometry analysis showed that the expression level of HLA recombinant proteins modified with Fc on the yeast surface was significantly higher than that of HLA recombinant proteins modified with LZ (Figure 8). This result confirms that C-terminal modification of the designed HLA fusion proteins, particularly with an AGA2 SP signal peptide to guide extracellular delivery and / or Fc modification, can effectively promote the expression of HLA extracellular domains in yeast.

[0239] The inventors then selected a peptide fragment that can specifically bind to both DR1 and DR4, namely influenza hemagglutinin (HA). 306-318(as shown in SEQ ID NO: 2) to verify yeast cell display of peptide / MHC-II trimers. Expression of the antigenic peptide and HLA-DR α / β heterodimers was driven by two sets of yeast shuttle vector plasmids. First, regardless of whether the peptide / MHC-II trimer was formed, since the antigenic peptide was expressed solely by the peptide-displaying plasmid, daughter yeast cells transformed with these plasmids were able to display the single-chain fusion antigenic peptide on the cell surface by fusing the peptide fragment to the C-terminus of the endogenous yeast adhesion receptor subunit Aga2p ( Figure 9 ). The vectors directing the expression of the single-chain fusion antigenic peptide gene used conventional yeast display methods (see, e.g., Boder & Wittrup, Yeast surface display for screening combinatorial polypeptide libraries, 1997). To successfully dimerize the selected representative HLA allele proteins and co-display them on the yeast surface via non-covalent binding (rather than direct display) with single-chain fusion antigen peptides, the inventors first deleted the gene encoding the Aga2p protein used for yeast HLA display from the aforementioned HLA display plasmid. This resulted in an HLA secretion vector that only secretes the HLA dimer structure without displaying the C-terminally enhanced HLA dimer backbone (Figures 2-7). This dimeric fusion protein is secreted from yeast cells independently of the single-chain fusion antigen peptide expression vector and then successfully binds to the antigen peptide within the cell, ultimately anchoring the peptide / HLA trimer structure on the cell surface (Figure 1). The vector directing the display of the single-chain fusion antigen peptide and the C-terminally enhanced HLA secretion vector were transformed into Saccharomyces cerevisiae EBY100 for protein expression and trimer co-display. Using dual antibody fluorescent labeling and flow cytometry detection, the inventors successfully demonstrated that the CODAAH system can display peptide / HLA trimers on the cell surface (Figure 9). Importantly, modifications to the HLA C-terminus, particularly the Fc modification, significantly increased the abundance and display of peptide / HLA trimers formed by DR1 on the yeast surface (expressed as mean or median fluorescence signal intensity (MFI), with a signal enhancement of more than 2-fold). This method can also enable the successful formation of peptide / HLA trimers from proteins of some HLA alleles that are difficult to co-display on the yeast surface (expressed as mean or median fluorescence signal intensity, with signals increasing from zero to significant levels).

[0240] Example 2: Construction of a display peptide library to achieve CODAAH system optimization and antigen peptide development

[0241] The co-display of peptides / HLA trimers by the CODAAH system depends on the modification and stabilization of the HLA-IIα / β heterodimer protein itself and on the preference for HLA-II specific antigen peptides.306-318 The amino acid sequence of HA was analyzed and a peptide variant library was designed. 306-318 Registered peptide sequence: YVKQNTLKL (as shown in SEQ ID NO: 20), in which the five amino acid residues that mainly bind to DR1 are randomly mutated, with the intention of screening out peptide variants with higher affinity to DR1 by flow cytometry sorting, so that more DR1 dimers can be displayed on the yeast surface by binding to peptide variants. The anchor residues that YVKQNTLKL binds to DR1 are mainly P1Y, P4Q, P6T, P7L, and P9L. The inventors used PCR primers carrying five degenerate codons (NNS) to amplify the insert fragment and double enzyme digestion to obtain a linearized empty plasmid. The high concentration of the carrier encoding HA 306-318 The insert fragment of the peptide variant sequence (as shown in SEQ ID NO: 46) and the linearized empty vector plasmid were transformed into yeast cells that can secrete and express DR1. The insert fragment and the linearized empty vector plasmid formed a plasmid displaying the peptide variant through homologous recombination in the daughter yeast cells, so that each yeast cell in the library can display at least one peptide variant and can simultaneously secrete DR1 molecules. The library constructed in this way has a capacity of >10 8 Flow cytometry sorting was used to screen for double positive peptide variants (signal represented by HA tag) and DR (signal represented by L243 monoclonal antibody), and the target yeast clone was obtained. After 4 rounds of enrichment and sorting, the progeny library (about 0.01% of the parent library, ~10 4The HA+ / DR+ double-positive signal of the yeast clone (the double-positive signal of the parent library) was significantly improved compared to the double-positive signal of the parent library (Figure 10). The sequencing results of the plasmids contained in the single clones of the sub-library showed that 90% of the clones carried peptide variants with base mutations (Figure 11). In the bacterial PCR sequencing results directly performed on the yeast clones, the designed degenerate codon positions mostly showed overlapping peak sequencing results (8 out of 10, except #1 and #4), indicating that the HA+ / DR+ double-positive single yeast cell contains multiple peptide variant display plasmids, and the peptide variants expressed by each plasmid are not exactly the same at the five anchor residue positions. It is further inferred that the dominant codon DNA of the peptide variants of yeast clones #5 and clone #7 is the same as the product corresponding to the dominant codon, and it cannot be confirmed that the peptide variant display recombinant plasmids contained in these two yeast clones are exactly the same. The inventors extracted the plasmids contained in the monoclonal yeasts numbered #1, #7, #9, and #10 and transformed them into E. coli. The results of plasmid extraction and sequencing from multiple selected E. coli verified this hypothesis: a single yeast cell can accommodate at least 3 peptide variant display plasmids, and the peptide variant expressed by each plasmid can bind to DR1 (Figure 12). In addition, the yeast PCR sequencing did not show overlapping peaks in the #1 clone, which indeed contained only one peptide variant (the sequencing results of the E. coli extracted plasmids #1-1 and #1-2 were exactly the same as the yeast #1 PCR sequencing results), and the #7, #9, and #10 clones that showed overlapping peaks also contained more than one peptide variant. This conclusion shows that the actual library capacity of the constructed parent library may be larger than the theoretical library capacity, and may even exceed it by an order of magnitude, approaching the industrial-grade library capacity 10 9 This also means that the number of peptide variants that can bind to DR1 in the HA+ / DR+ double-positive sub-library can reach 10 5 , among which up to 90% of the peptide variants may contain actual amino acid mutations at the anchoring site, and have a promoting effect on improving DR1 affinity, thereby facilitating the co-display of the peptide and DR1 trimer formed by the CODAAH system.

[0242] The effect of constructing library-preferred peptide variants to promote peptide / HLA trimer co-display can even improve the application of the CODAAH system in other related peptide / DR co-displays. For example, with wild-type HA 306-318 Compared with (Figure 9), peptide variant #1 not only improves its ability to bind to DR1, but also improves its ability to bind to DR4 in DR4-secreting yeast transformant strains (Figure 13). And the important point is that compared with DR4-LZ, the Fc modification in the DR4-Fc fusion protein structure is obviously more conducive to the detection of relatively high levels of DR4 protein on the surface of transformed yeast cells by flow cytometry (Figure 13), which once again verifies the optimal effect of C-terminal Fc modification on stabilizing HLA-IIα / β heterodimers. 306-318The method of discovering DR1 neoantigen peptides using a variant library is similar. Using peptide variant #1 as the wild type, a variant library #1 can be constructed as a method for discovering DR4 neoantigens. It is worth mentioning that through blast analysis of peptide variant #1, the inventors found that the five DR binding anchor residues P1Y, P4L, P6P, P7A, and P9A of the registered peptide sequence of peptide variant #1 (YVKLNPAKA (as shown in SEQ ID NO: 25)) are exactly the same as the five anchor residues of the potential DR registered peptide sequence (YIKLNPADA (as shown in SEQ ID NO: 38)) of the Escherichia coli NADH-quinone oxidoreductase subunit NuoG. This discovery can guide the prediction of neoantigen peptides derived from Escherichia coli and possible related immune responses that may be produced by DR1 or DR4 positive people. In summary, the above conclusions demonstrate the important role of the CODAAH system in the application of high throughput and high efficiency in peptide optimization and discovery of HLA neoantigen peptides.

[0243] Materials and methods used in Examples 1 and 2

[0244] Unless otherwise specified, all materials and reagents used were commercially available. All experimental methods used were conventional methods unless otherwise specified.

[0245] Construction and electroporation of yeast co-display shuttle vectors

[0246] The entire gene expression cassette (GAL1-10 / / AGA2-HA / / scFv 4-4-20 / / MFαTerm.) was excised from pCT302 using KpnI and SacI and then subcloned into the yeast shuttle vector pRS315, which had been partially digested with KpnI / SacI, to form a yeast surface display plasmid with the LEU nutritional marker gene. The DNA chain encoding scFv 4-4-20 in the original plasmid was then replaced with an oligonucleotide DNA chain encoding an antigenic peptide (e.g., influenza virus hemagglutinin residues HA306-318: SEQ ID NO: 2), a short spacer (GGGS), and a V5 epitope tag to construct a vector for displaying Aga2-single-chain antigen peptide fusion protein.

[0247] The plasmids ptDR4-LZ and ptDQ6-LZ (Liu, Jiang, & Mellins, Yeast display of MHC-II enables rapid identification of peptide ligands from protein antigens (RIPPA), 2021) can be used to display C-terminally LZ-enhanced HLA-DR4 and HLA-DQ6 ectodomain heterodimers in yeast, respectively. To create a vector for displaying C-terminally LZ-enhanced HLA-DR1 ectodomain heterodimers in yeast, the β gene HLA-DRB1*04:01 in ptDR4-LZ was replaced with HLA-DRB1*01:01. To create a vector for secreting C-terminally enhanced HLA ectodomain heterodimers in yeast, the nucleic acid sequences encoding the AGA2 and HA tags downstream (3'-end) of the DRβ or DQ6α genes in the three plasmids used for yeast HLA-II display were replaced with in-frame stop codons. The two sets of vectors thus obtained can be used to display or secrete three representative HLA fusion proteins, DR1, DR4, and DQ6, with enhanced C-terminal leucine binding domain modifications (see Figures 2-4). Furthermore, through molecular cloning, the first and second leucine zipper fragments downstream (3'-end) of the HLA α and β genes in each of the two sets of three vectors (one set of three display vectors and the other set of three secretion vectors) were replaced with antibody crystallizable fragments. Thus, the resulting two sets of three new vectors (one set of three display vectors and the other set of three secretion vectors) can direct the expression of DR1, DR4, and DQ6 fusion proteins with enhanced C-terminal crystallizable antibody constant domain modifications (see Figures 5-7). Next, the nucleic acid sequence encoding the AGA2SP signal peptide (shown in SEQ ID NO:39) was cloned into the N-terminus of the Fc-modified DRβ chain or DQα chain, replacing the original nucleic acid sequence encoding the SPP SP signal peptide (shown in SEQ ID NO:45).

[0248] TRP+ vector plasmids for secreting or displaying HLA-II, along with LEU+ vectors for expressing single-chain antigen peptide fusion proteins, or each plasmid alone, were transformed into the Saccharomyces cerevisiae strain EBY100 (GAL1-AGA1:URA3 ura3-52trp1 leu2Δ1 his3Δ200 pep4:HIS2 prb1Δ1.6R can1 GAL). Electroporation was performed using a MicroPulser electroporator (BioRad), following the basic protocol described in the BioRad MicroPulser manual. Single yeast colonies harboring the TRP+ and / or LEU+ vectors formed visible single colonies on solid agar plates containing tryptophan and / or leucine deficiency after 2-3 days of incubation at 30°C. The culture medium SD-SCAA contains 2% (wt / vol) glucose, 0.67% (wt / vol) yeast nitrogen base without amino acids, appropriate amount of amino acid (Trp- and / or Leu- and optionally Ura-) deletion supplement mixture (Clontech), 38 mM Na2HPO4, 62 mM NaH2PO4, pH 6.

[0249] Preparation of co-display yeast for flow cytometry

[0250] A single yeast colony was used to inoculate 2 ml of SD-SCAA medium and cultured in a shaking incubator at 30°C until the number of cells reached 2.5-5.0 × 10 7 To induce protein expression promoted by GAL1-10, cells were collected by centrifugation at a density of 10 cells / mL (OD600 was 2.5-5.0). 7 After induction at 30°C for 16-18 hours, 10 cells were harvested for each sample by centrifugation. 6Cells were used for immunofluorescence labeling. When protein stripping was required, induced cells were first washed 1-2 times with 400 μL Tris-buffered saline (137 mM NaCl, 20 mM Tris-Cl, pH 7.6), then gently shaken for 24 hours at 4°C in 20 μL reducing buffer [50 mM Tris-Cl pH 8.0, 1 mM DTT (Sigma; added before use)], or incubated in 20 μL Factor Xa buffer (100 mM NaCl, 2 mM CaCl2, 20 mM Tris-Cl, pH 8) with 20 μg / mL Factor Xa protease (New England Biolabs) at 23°C for at least 48 hours. Before primary labeling, cells were pelleted by centrifugation and washed at least once with 400 μL ice-cold PBS + 1% BSA. The cell pellet was resuspended in 25 μL of PBS + 1% BSA containing mouse anti-DR monoclonal antibody L243 (BD ​​Biosciences) or anti-DQ monoclonal antibody SPV-L3 (BD ​​Biosciences) and rabbit anti-HA polyclonal antibody (Sigma) and incubated at room temperature for 30 minutes, followed by incubation on ice for 10 minutes. After centrifugation to remove the primary reagents, the cells were washed again with ice-cold PBS + 1% BSA and resuspended in 40 μL of PBS + 1% BSA containing highly cross-adsorbed secondary antibodies (Thermo Fisher): Alexa Fluor 647 goat anti-mouse IgG (H+L) (1:80) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) (1:80) and incubated on ice for 40 minutes. Finally, the cells were washed with ice-cold PBS + 1% BSA and resuspended in 500-700 μL of PBS + 1% BSA for flow cytometry analysis. For simultaneous detection of HA-tag and V5-tag flanking peptides, mouse anti-V5 mAb (Thermo Fisher) was used at a dilution of 1:30 instead of mAb L243 as the primary labeling reagent, while other labeling steps remained unchanged.

[0251] Construction of a Peptide Variant / HLA Trimer Co-display Yeast Library and Single-Clone Isolation and Analysis

[0252] To make the yeast in the library display HA 306-318 The peptide variants are preferably peptide variants that can bind to DR1. The inventors designed the following primers:

[0253] Among them, P0 is used in 5 HA 306-318Random mutations were introduced at the peptide anchor residues. P1 and P2 were used to amplify gene inserts carrying the peptide mutants, and homologous sequences to the linearized vector were added to the ends of the fragments. The linearized peptide display vector was obtained by double digestion with XmaI / NheI. After centrifugation, concentration, and drying, a total of 50 μg of insert and linearized vector (1:1 mass ratio) were electroporated into 800 μl of yeast competent cells secreting DR1 to obtain the yeast library for co-display. Immunofluorescence labeling of the yeast library was performed using the same amplification method as described above for the yeast strain. Yeast cells co-labeled with the HA tag and DR were sorted on a FACSAria IIu flow cytometer. The sorted sub-libraries were re-amplified in SD medium and then induced for expression in SG medium for flow cytometric analysis or further sorting. A sample of the final sorted and amplified sublibrary is plated on agarose for single-clone yeast PCR and gene sequencing. Yeast plasmid extraction and E. coli transformation are then performed, followed by plasmid extraction and gene sequencing analysis of multiple E. coli strains corresponding to the yeast strain. Sequencing-verified peptide variant display plasmids are re-transformed into yeast competent cells secreting DR1 or DR4 for trimer co-display verification. Verification methods can refer to flow cytometry analysis and the quantitative analysis methods described below.

[0254] Flow cytometric detection and quantification of relative binding

[0255] At least 10,000 cell events were collected for each sample and gated by forward and side scatter. The flow cytometers used included FACSCalibur and FACSAriaIIu (BD Biosciences). Flow cytometric data of yeast strains co-displayed with peptide / HLA trimers were analyzed using FlowJo software (BD). 306-318 The level of display of ) on the yeast surface is proportional to the background-corrected mean fluorescence intensity value and normalized to the background intensity, expressed as cMFI.

[0256] Where MFI(HA-) and MFI(HA+) represent the mean fluorescence intensity of HA-tag-conjugated Alexa Fluor 488 emitted by negative and positive cell populations, respectively, calculated using FlowJo. Analysis of peptide display levels on the yeast surface using anti-V5-tag staining yields comparable cMFI values ​​to those described above.

[0257] HLA display levels that are dependent on peptide binding can be accurately calculated by using the normalized, background-corrected fluorescence versus anti-DR staining and peptide display levels (DR-ratio) (anti-DQ staining is calculated similarly).

[0258] Wherein, (+) and (-) represent the yeast with co-display and yeast with peptide display only, respectively. MFI(DR) and cMFI represent the mean fluorescence intensity of Alexa Fluor 647 and HA conjugated to DR. 306-318 Display levels on the surface of the corresponding yeast strains. Normalization minimizes inter-experimental variation due to laser power output, detector amplification, and other cytometer parameters.

[0259] Example 3: This method effectively increases the display of "empty" MHC-II complexes

[0260] In the endoplasmic reticulum of professional human antigen-presenting cells (APCs), the peptide-binding groove of nascent MHC-II (or human leukocyte antigen, HLA-II) is typically occupied by the partner invariant chain (Ii), which stabilizes the HLA-II protein structure and prevents mispairing between HLA-II and interfering intracellular peptides. Ii can be trimmed by proteolytic enzymes to produce CLIP peptides. CLIP peptides continue to bind HLA-II with lower affinity until higher-affinity antigenic peptides displace CLIP through a peptide exchange process. This process often occurs in the MHC-II class II compartment (MIIC), where peptide exchange is typically catalyzed and regulated by HLA-DM. These immunological data indicate that MHC-II proteins with an "empty" peptide-binding groove are unstable, a finding that has been confirmed in numerous protein expression systems. To effectively increase yeast expression of "empty" MHC-II, the inventors used molecular cloning techniques to link one chain (α or β) of a given MHC-II allele to a yeast surface protein, while allowing the other chain (α or β) to be secreted from the same yeast cells as a soluble component. The inventors modified the C-termini of the α / β chains to carry the amino acid sequence of the crystallizable fragment of an antibody heavy chain (FcAB, or simply Fc in the description of HLA-II fusion proteins). This amino acid sequence effectively promotes pairing of the soluble secretory chain with other surface-anchored chains, significantly increasing the expression and / or copy number of the MHC-II complex.

[0261] result

[0262] The inventors used HLA-DR1 (HLA-DRA*01:01 / HLA-DRB1*01:01, as shown in SEQ ID NO:40 and SEQ ID NO:41), HLA-DR4 (HLA-DRA*01:01 / HLA-DRB1*04:01, as shown in SEQ ID NO:40 and SEQ ID NO:42), and HLA-DR15 (HLA-DRA*01:01 / HLA-DRB1*15:01, as shown in SEQ ID NO:40 and SEQ ID NO:81) as representative DR alleles; and used HLA-DQ6 (HLA-DQA1*01:02 / HLA-DQB1*06:02, as shown in SEQ ID NO:43 and SEQ ID NO:44) as a representative DQ allele. In yeast shuttle vectors constructed to express DR1, DR4, DR15, or DQ6 complexes, the bidirectional GAL1-10 promoter directed simultaneous expression of both the α and β chains. Compared to previously used leucine zipper (LZ) motifs to promote dimerization, HLA-II α / β chains with C-terminal FcAB modifications were more likely to form "empty" HLA complexes (Figure 14).

[0263] The inventors created new yeast strains by transforming the successfully constructed yeast shuttle vectors into the parental Saccharomyces cerevisiae strain EBY100 (Boder & Wittrup, 1997). After inducing protein expression, the inventors performed immunofluorescence labeling on the yeast cells and flow cytometry analysis. The detection antibodies are a mouse monoclonal antibody L243 and a mouse monoclonal antibody that recognizes the spatial epitope of the correctly folded HLA-II αβ protein complex. Flow cytometry measurements showed that FcAB-modified HLA-IIα / β expression levels on the yeast surface were generally higher than those on LZ-modified HLA-IIα / β, including DR1, DR15, and DQ6 (MFI_Fc > MFI_LZ). Specifically, the HLA-II-positive fluorescence intensity signals of DR15 and DQ6 increased significantly by 172.7% and 86.6% (MFI_Fc - MFI_LZ) / (MFI_LZ - MFI_BC) x 100%, respectively (Figure 14). This increased expression level is attributed to the high affinity and binding specificity between the FcA and FcB binding domains.

[0264] Example 4: This method can be further optimized and increased to display “empty” MHC-II complexes

[0265] introduce

[0266] Eukaryotic cells typically secrete proteins using a signal peptide (SP) at the N-terminus of the amino acid polypeptide sequence. MHC-II duplexes displayed in yeast also require a similar signal peptide. Previously constructed yeast shuttle vectors expressing DR1, DR4, DR15, or DQ6 complexes used syn-pre-pro synthetic signal peptides at the N-terminus of both the α and β chains. To further increase the amount of yeast surface display of the "empty" HLAα / β complex, the inventors replaced the N-terminal syn-pre-pro synthetic signal peptide of the yeast display chain with the yeast endogenous AGA2 signal peptide (Figure 15).

[0267] result

[0268] After inducing protein expression, the inventors performed immunofluorescence labeling and flow cytometry analysis on the newly constructed yeast cells. Due to the use of the AGA2 SP, the expression levels of FcAB-modified DR1 or DR4α / β on the yeast surface were significantly increased, significantly higher than the expression levels of LZ-modified HLA-IIα / β on the yeast surface (MFI_Fc > MFI_LZ). The positive fluorescence intensity signal increased by 146.6% and 96.1% (MFI_Fc - MFI_LZ) / (MFI_LZ - MFI_BC) x 100%, respectively (Figure 15).

[0269] In summary, FcAB modification combined with the optimization of signal peptides enables the correct assembly of all "empty" MHC-II proteins on the yeast surface and significantly improves the display level, which lays a solid foundation for improving the sensitivity and accuracy of MHC-II peptide ligand identification.

[0270] Example 5: This method effectively improves the sensitivity and accuracy of identifying MHC-II peptide ligands

[0271] Identifying peptide ligands for MHC-II involves both computational prediction and experimental validation. Because artificial intelligence and machine learning are still in their developmental stages, computational prediction algorithms still rely heavily on highly accurate experimental data. Currently, experimental methods for identifying peptide ligands for MHC-II fall into two categories. One uses mass spectrometry to quantify peptides eluted from MHC-II molecules immunoprecipitated from lysed cells (EL). The other involves detecting the binding affinity of synthetic peptides to recombinant MHC-II proteins (BA). To identify peptides that bind to proteins expressed by a specific MHC-II allele, the optimal approach for both experimental methods is to generate cell lines expressing only that allele rather than using primary human cells. This is because primary human cells often express multiple MHC-II alleles and are dominantly co-expressing alleles from the three human leukocyte antigens (HLA) groups: HLA-DR, HLA-DQ, and HLA-DP. Furthermore, both methods require purification or enrichment of the expressed MHC-II protein before identifying peptide ligands. Establishing a cell line expressing a single MHC-II allele and purifying MHC-II proteins typically takes 4-6 months, severely limiting the speed of research and development. Furthermore, most EL methods are qualitative and have a higher false-positive rate than BA methods. In comparison, the identification and determination of peptide ligands using "empty" MHC-II displayed on the yeast surface can take as little as one week and achieve both qualitative and quantitative results. More importantly, the higher the yield and copy number of "empty" MHC-II proteins displayed on the yeast surface, the more helpful it is in improving the sensitivity and accuracy of identifying MHC-II peptide ligands. Therefore, FcAB-modified "empty" MHC-II proteins offer significant advantages in this application area.

[0272] While physiological peptide loading occurs in the acidic MIIC (pH 5) at human body temperature (37°C), "empty" MHC-II displayed on the yeast cell surface can bind specific target peptides under a wide range of conditions, such as pH 5.0 or pH 7.4 and at 30°C or 37°C. Here, the target peptide is biotinylated (indicator peptide) for flow cytometric detection and quantification. Kinetic evidence indicates that dynamic equilibrium between the target peptide and the "empty" MHC-II displayed on the yeast surface generally occurs within 15–20 hours. Therefore, pH 5 for 20 hours represents the preferred condition for comparing the levels of HLA-II-Fc and HLA-II-LZ yeast display and the sensitivity and accuracy of their peptide binding.

[0273] result

[0274] To target the “empty” HLA-DR1 complex protein displayed on the yeast surface, the inventors used influenza hemagglutinin (HA) 306-318The peptide is the specific target antigen peptide corresponding to HLA-DR1. 306-318 Comparative experiments using the presence of an indicator peptide revealed that not only was the expression level (displayed amount) of DR1-Fc detected on the yeast surface much higher than that of DR1-LZ, but the level of indicator peptide bound was also significantly higher than that of DR-LZ (Figure 16). This demonstrates that the apparent activity of "empty" HLA-IIα / β modified with FcAB on the yeast surface is higher than that of LZ-modified proteins (i.e., the parameter: peptide binding capacity per unit of yeast). In other words, a lower concentration of indicator peptide staining yeast expressing FcAB-modified HLA-IIα / β can achieve the same fluorescence signal intensity (expressed as MFI) as that obtained with a higher concentration of indicator peptide staining yeast expressing LZ-modified HLA-IIα / β (Figures 16 and 17). This demonstrates that the yeast strains generated using this method have higher sensitivity than other yeast strains for peptide ligand identification. This increased sensitivity also means that affinity constants (e.g., the apparent equilibrium dissociation constant, KDapp) determined by varying indicator peptide concentrations are more accurate, more closely approximating those determined by traditional BA methods. For example, Bio-HA assay using DR-Fc display yeast 306-318 The affinity KDapp is 6.81 μM, which is significantly closer to the HA determined by the traditional BA method than 15.14 μM (DR-LZ determination value). 306-318 KD of the order of ∼100 nM ( FIG. 17 ).

[0275] In further experiments, the inventors also used non-biotinylated HA 306-318 (competing peptide) and biotinylated Bio-HA 306-318 (Indicated peptide) was tested for competitive binding with MHC-II. The experimental results showed that the yeast-displayed DR-Fc bound to Bio-HA 306-318 HA 306-318 The signal-to-noise difference of the competition is significantly greater than that of yeast-displayed DR-LZ (ΔMFI = 18.5 VS ΔMFI = 3.81) (Figure 18). In addition, the half-inhibitory concentration (IC50) measured using FcAB-modified "empty" DR1α / β expressed on the yeast surface is more accurate than the IC50 measured using the LZ-modified complex, because when the concentration of the competing peptide is higher than 50 μM, it is almost impossible to distinguish the positive signal of the indicator peptide from the background signal on the yeast surface displaying DR-LZ, resulting in a significant increase in data fitting error (Figure 19). This comparison result once again proves that the yeast strain created by this method has higher sensitivity and accuracy in the application scenario of identifying peptide ligands than other yeast strains.

[0276] Materials and methods for Examples 3-5

[0277] Material

[0278] The indicator peptides: Bio-HA306-318 (biotin-PKYVKQNTLKLAT), Bio-aI (negative control, DQ2.5 binding peptide) and competitor peptides were synthesized by GenScript. Monoclonal antibodies (mAbs) included mouse anti-DRαβ (clone L243), mouse anti-DQαβ (clone ), Alexa Fluor 647-conjugated streptavidin, and highly cross-adsorbed secondary antibodies including Alexa Fluor 488 goat anti-mouse IgG (H+L) and Alexa Fluor 647 goat anti-mouse IgG (H+L) were purchased from Thermo Fisher Scientific.

[0279] method

[0280] Creation of yeast display vector

[0281] To construct "empty" DR4-Fc and "empty" DQ6-Fc display plasmids, the Fos or Jun leucine zipper dimerization motif at the C-terminus of the α or β chains in ptDR4-LZ(TRP+) and ptDQ6-LZ(TRP+) used previously (Liu et al., 2021) was replaced with the FcA or FcB domain (shown in SEQ ID NOs: 11-19, with sequences selected in the examples shown in SEQ ID NOs: 14 and 15). DR4 can be replaced with DR1 or DR15 to construct yeast display vectors for DR1-Fc or DR15-Fc.

[0282] Yeast transformation, culture, and protein expression

[0283] The plasmid carrying the tryptophan nutritional marker gene (TRP+) was transformed into the yeast parent strain EBY100 (URA+, TRP-) by electroporation according to the BioRad MicroPulser manual protocol. After culturing in a 30°C incubator for 2 days, a single yeast colony appeared on a solid agar plate containing a tryptophan-deficient medium consisting of SD-CAA (2% w / v glucose, 0.67% w / v yeast nitrogen base without amino acids, 0.062% w / v Ura / Trp-deficient casamino acids, 38 mM Na2HPO4, 62 mM NaH2PO4, pH 6.0). A single yeast colony was then used to inoculate 2 ml of SD-CAA liquid medium and cultured overnight at 30°C with shaking at 225 rpm until the OD 600 To induce GAL1-10 driven protein expression in yeast, 10 7After induction for 18 hours at 30°C, sufficient yeast cells were harvested by centrifugation at 2,500 g for 3 minutes, washed, and prepared for analysis of protein expression or peptide binding.

[0284] Immunofluorescence staining and flow cytometry

[0285] The expression and display levels of "empty" MHC-II were assessed by flow cytometry using immunofluorescence labeling. Briefly, galactose-induced yeast cells were first incubated with antibodies containing either mouse mAb L243 (for DR) or The cells were labeled with a primary mAb (for DQ) or other tag-tagged antibodies (approximately 10 μg / ml for each mAb) for 30 minutes at room temperature (RT), followed by 30 minutes on ice. The cells were then washed with 300 μl of ice-cold PBS + 1% w / v bovine serum albumin (BSA) and incubated on ice for 1 hour with a highly cross-adsorbed secondary antibody, AlexaFluor 488 goat anti-mouse IgG (H+L) or AlexaFluor 647 goat anti-mouse IgG (H+L) (1:100 dilution), or other secondary antibodies of other origin. Following labeling, yeast cells were analyzed on a flow cytometer to detect fluorescent signals corresponding to expression of MHC-II proteins or epitope tags. No fewer than 20,000 cells were collected for each sample. Flow cytometric data were analyzed using FlowJo software (BD Biosciences).

[0286] Target antigen peptide binds to the "empty" MHC-II on yeast

[0287] 6 × 10 cells expressing “empty” MHC-II were collected by centrifugation at 2,500 g for 3 minutes. 5 Galactose-induced yeast cells were prepared and resuspended in 40 μl of the following solutions: citrate buffer (40 mM citric acid and sodium citrate, pH 5.0, 150 mM sodium chloride) and phosphate-buffered saline (PBS; pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4). The indicated peptides were added to the solution and incubated under the desired conditions. To determine the kinetics of peptide binding, 6 × 10 5Yeast cells induced with galactose were resuspended in 40 μl of 40 mM citrate buffer (pH 5.0) and incubated with the indicated peptides at various time points. The cells were then harvested by centrifugation at 2,500 g for 3 minutes for flow cytometric analysis. To determine the apparent affinity of MHC-II peptide binding on the yeast cell surface, yeast cells displaying "empty" MHC-II were incubated with varying concentrations of the indicated peptides in 40 mM citrate buffer (pH 5.0) at 30°C for 20 hours. The reaction tubes were sealed with parafilm before incubation to prevent changes in culture volume that could affect the final concentration of the peptide in time course or concentration titration studies. After incubation, the yeast cells were washed twice with 300 μl of ice-cold PBS + 1% BSA and then stained with streptavidin-AF647 diluted 1:200 in 50 μl of PBS + 1% BSA for one hour on ice. The cells were then washed twice with 300 μl of ice-cold PBS + 1% BSA and finally resuspended in 300 μl of ice-cold PBS + 1% BSA for flow cytometric analysis. To simultaneously detect cell surface MHC-II proteins and indicator peptides, cells that bound the indicator peptides were first incubated on ice with mouse mAb L243 (for DR) or (For DQ) staining was performed for 30 minutes, followed by double labeling with highly cross-adsorbed Alexa Fluor 488 goat anti-mouse IgG (H+L) and streptavidin-AF647 in PBS+1% BSA on ice for one hour. Peptide binding was quantified as the de-noised streptavidin staining signal (MFI SA,indicator -MFI SA,neg ) and plotted against incubation time or peptide concentration to determine kinetic or thermodynamic parameters. To determine the apparent equilibrium dissociation constant (KDapp) for binding of the indicated peptides to "empty" MHC-II molecules on the yeast cell surface, the data were fitted using nonlinear regression using a site-specific binding equation in Graphpad Prism.

[0288] Peptide competition assay using yeast displaying MHC-II

[0289] 6×10 5 Galactose-induced yeast were incubated with 10 μM indicator peptide in the presence of various concentrations of competitor peptide at pH 5.0 and 30°C for 20 hours to determine the appropriate competitor concentration for the competition assay. After incubation, yeast cells were washed with PBS + 1% BSA, stained with streptavidin-AF647 and analyzed by flow cytometry as described above. The percentage of binding in the presence of competitor peptide was quantified as [(MFI withcompetitor -background) / (MFI withoutcompetitor= -background)] x 100% and plotted against competitor concentration to determine the half inhibitory concentration (IC50). Data were fitted using nonlinear regression using a one-point fit log IC50 equation in Graphpad Prism.

[0290] The sequences used in the above examples of the present application are shown in the following sequence table. It should be understood that the following sequences are merely exemplary sequences of the embodiments of the present application, and are not intended to limit the present application.

[0291] Sequence Listing

Claims

1. A cell comprising a major histocompatibility complex (MHC) and a single chain domain, wherein the complex comprises an α chain and a β chain, wherein: The α chain is linked to a first protein binding domain to form a first fusion protein, The beta chain is linked to a second protein binding domain to form a second fusion protein, The single-chain domain non-covalently binds to the α chain and the β chain, the first fusion protein binds to the second fusion protein to form the complex, and the complex and the single-chain domain form a trimer, wherein, The first protein binding domain binds to the second protein binding domain to enhance or promote the formation of a trimer formed between the complex and the single-chain domain.

2. The cell according to claim 1, wherein The first protein binding domain is non-covalently bound to the second protein binding domain; or the first protein binding domain is covalently linked to the second protein binding domain, and the covalent bond is preferably a disulfide bond.

3. The cell according to claim 1, wherein The cell is a yeast cell.

4. The cell according to claim 1, wherein The single-chain domain is bound to the surface of the cell by covalent fusion with the molecule on the cell surface. 5 . The cell according to claim 4 , wherein the α chain or the β chain is bound to the molecule on the cell surface by non-covalent binding to the single-chain domain.

6. The cell of claim 4, wherein the molecule is a protein.

7. The cell of claim 6, wherein the protein is endogenous to the cell.

8. The cell of claim 6, wherein the protein is Aga2p, a-lectin, α-lectin, flocculin, Cwp1p, Cwp2p, or Tip1p.

9. The cell of claim 6, wherein the protein is Aga2p. 10 . The cell according to claim 9 , wherein the amino acid sequence of Aga2p is as shown in SEQ ID NO:

1.

11. The cell according to claim 1, wherein the single-chain domain is a peptide segment of 9 to 30 amino acids in length, and the single-chain domain contains at least one continuous register peptide of 9 amino acids in length that can be specifically recognized by the complex or a registered peptide variant that can be bound by the complex, preferably the register peptide or its variant is selected from the amino acid sequence shown in any one of SEQ ID NOs: 20-38.

12. The cell according to claim 11, wherein the amino acid sequence of the single-chain domain is shown in any one of SEQ ID NOs: 2-6 or 46-77.

13. The cell according to claim 1, wherein The first protein binding domain is a first leucine zipper domain, and the second protein binding domain is a second leucine zipper domain, wherein the first leucine zipper domain and the second leucine zipper domain that form a complete leucine zipper can be used interchangeably in the first fusion protein and the second fusion protein; or The first protein binding domain is an FcA domain, and the second protein binding domain is an FcB domain, wherein the FcA domain and the FcB domain that form the FcAB dimer can be used interchangeably in the first fusion protein and the second fusion protein.

14. The cell according to claim 13, wherein The amino acid sequence of the first leucine zipper domain is shown in SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10; Correspondingly, the amino acid sequence of the second leucine zipper domain is shown in SEQ ID NO:8 or SEQ ID NO:7 or SEQ ID NO:10 or SEQ ID NO:

9.

15. The cell according to claim 13, wherein The FcA domain and the FcB domain are identical or different amino acid sequences, preferably selected from any one of the amino acid sequences shown in SEQ ID NOs: 11-19; Preferably, the Fc A domain and FcB domain are capable of increasing the expression display amount and / or copy number of the complex; Preferably, the FcA domain comprises only the first CH3 domain, and the FcB domain comprises only the second CH3 domain; or Preferably, the FcA domain comprises a first CH2 domain and a first CH3 domain, and the FcB domain comprises a second CH2 domain and a second CH3 domain; It is further preferred that the first CH2 domain and the first CH3 domain of the first protein-binding domain are connected via a linker, and the second CH2 domain and the second CH3 domain of the second protein-binding domain are connected via a linker.

16. The cell according to claim 15, wherein Either or both of the FcA domain and the FcB domain comprise one or more amino acid modifications that achieve any one, two or three of the following: (1) enhancing or stabilizing the FcA domain and / or the FcB domain itself ("FcS"), (2) enhancing or stabilizing the covalent or non-covalent binding between the FcA domain and the FcB domain ("FcM"), (3) reducing or avoiding mispairing of the α chain and the β chain caused by mispairing of the FcA domain with the FcB domain ("FcN").

17. The cell according to any one of claims 1, wherein The first protein binding domain is connected to the C-terminus of the α chain, and the second protein binding domain is connected to the C-terminus of the β chain. Preferably, the signal peptide for initiating the expression of the first fusion protein and the second fusion protein is SPP SP or AGA2 SP. Further preferably, the amino acid sequence of the AGA2 SP is as shown in SEQ ID NO:39, and the amino acid sequence of the SPP SP is as shown in SEQ ID NO:

45.

18. The cell according to claim 1, wherein The complex is an MHC class I molecule or an MHC class II molecule, and preferably the complex is an HLA class I molecule or an HLA class II molecule.

19. The cell according to claim 18, wherein The α chain is encoded by HLA-DRA*01 or other alleles of the HLA-DRA family, and the β chain is encoded by HLA-DRB1*01 or HLA-DRB1*03 or HLA-DRB1*04 or HLA-DRB1*15 or other alleles of the HLA-DRB family; or The α chain is encoded by HLA-DQA1*01 or HLA-DQA1*03 or HLA-DQA1*05 or other alleles of the HLA-DQA1 family, and the β chain is encoded by HLA-DQB1*02 or HLA-DQB1*03 or HLA-DQB1*05 or HLA-DQB1*06 or other alleles of the HLA-DQB1 family; or The α chain is encoded by HLA-DPA1*01:03 or HLA-DPA1*02:02 or other alleles of the HLA-DPA1 family, and the β chain is encoded by HLA-DPB1*01:01 or HLA-DPB1*02:01 or HLA-DPB1*04:01 or Encoded by HLA-DPB1*04:02 or other alleles of the HLA-DPB1 family.

20. A nucleic acid encoding a major histocompatibility complex (MHC), wherein the complex comprises an α chain and a β chain, wherein the α chain is connected to a first protein binding domain, and the β chain is connected to a second protein binding domain, wherein the α chain and the β chain are non-covalently bound to a single-chain domain, and the first protein binding domain binds to the second protein binding domain to enhance or promote the formation of a trimer formed between the complex and the single-chain domain.

21. The nucleic acid according to claim 20, wherein the major histocompatibility complex encoded by the nucleic acid is the major histocompatibility complex (MHC) according to any one of claims 1 to 19.

22. A method for preparing a major histocompatibility complex (MHC), the method comprising: Transforming a cell with the nucleic acid of claim 20 or 21 and a nucleic acid encoding a single-chain domain, And culturing the cell under conditions where the complex according to any one of claims 1 to 19 is expressed.

23. The method of claim 22, wherein the cell is a yeast cell.

24. A method for identifying a peptide that binds to a major histocompatibility complex (MHC), the method comprising: i) displaying the single-chain domain library in a cell according to any one of claims 1 to 19, and ii) detecting the trimer on the surface of a monoclonal cell in a cell library displaying the single-chain domain according to any one of claims 1 to 19, thereby identifying the binding peptide of the complex according to any one of claims 1 to 19.

25. The method of claim 24, wherein the single-chain domain comprises a peptide, or a mutant of a peptide, or a library of mutants of a peptide, or a mixture of peptides.

26. The method of claim 24 or 25, wherein the peptide, or a mutant of a peptide, or a library of mutants of a peptide, or a mixture of peptides non-covalently binds to the complex.

27. A registered peptide or variant thereof capable of binding to the major histocompatibility complex (MHC), wherein: The registered peptide or variant thereof is selected from the amino acid sequence shown in any one of SEQ ID NOs: 20-38.

28. A cell comprising a major histocompatibility complex (MHC), wherein: The complex comprises an α chain and a β chain, wherein The α chain is linked to the first protein binding domain to form a first fusion protein, The β chain is connected to a second protein binding domain to form a second fusion protein, The first fusion protein binds to the second fusion protein to form the complex, The first protein binding domain and the second protein binding domain are used to enhance the binding of the α chain and the β chain to form the MHC.

29. The cell according to claim 28, wherein The cell is a yeast cell.

30. The cell according to claim 28, wherein The complex binds to the surface of the cell by linking the α chain or the β chain to a molecule on the surface of the cell.

31. The cell according to claim 30, wherein The additional α chain or the additional β chain in the complex binds to the molecule on the cell surface by non-covalently binding to the β chain or the α chain.

32. The cell according to claim 30, wherein The molecule is a protein.

33. The cell according to claim 32, wherein The protein is endogenous to the cell.

34. The cell according to claim 32, wherein The protein is Aga2p, a-lectin, α-lectin, flocculin, Cwp1p, Cwp2p or Tip1p.

35. The cell according to claim 32, wherein The protein is Aga2p.

36. The cell according to claim 35, wherein The amino acid sequence of Aga2p is shown in SEQ ID NO:

1.

37. The cell according to claim 28, wherein The first protein binding domain non-covalently binds to the second protein binding domain; or The first protein binding domain is covalently bound to the second protein binding domain, and preferably the covalent binding is through a disulfide bond.

38. The cell according to claim 28, wherein The first protein binding domain and the second protein binding domain form an FcAB domain; Preferably, the first protein binding domain is an FcA domain or an FcB domain, and the second protein binding domain is an FcB domain or an FcA domain, wherein, It is further preferred that the FcA domain and FcB domain can increase the expression level and / or copy number of the complex.

39. The cell according to claim 38, wherein The FcA domain only comprises the first CH3 domain, and the FcB domain only comprises the second CH3 domain; or The FcA domain comprises both a first CH2 domain and a first CH3 domain, and the FcB domain comprises both a second CH2 domain and a second CH3 domain; It is further preferred that the first CH2 domain and the first CH3 domain of the first protein-binding domain are connected via a linker, and the second CH2 domain and the second CH3 domain of the second protein-binding domain are connected via a linker.

40. The cell according to claim 39, wherein The FcA domain and the FcB domain are arbitrarily selected from any one of the amino acid sequences shown in SEQ ID NOs: 11-19 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 11-19.

41. The cell according to claim 39 or 40, wherein Either or both of the FcA domain and the FcB domain comprise one or more amino acid modifications, The modification can achieve any of the following: enhance or stabilize the FcA domain and / or the FcB domain itself ("FcS"), enhance or stabilize the covalent or non-covalent binding between the FcA domain and the FcB domain ("FcM"), The mispairing of the α chain and the β chain caused by the mispairing of the FcA domain with the FcB domain is reduced or avoided ("FcN").

42. The cell according to any one of claims 28 to 41, wherein The first protein binding domain is connected to the C-terminus of the α chain, and the second protein binding domain is connected to the C-terminus of the β chain. Preferably, the signal peptide for initiating the expression of the first fusion protein and the second fusion protein is SPP SP or AGA2 SP. Further preferably, the amino acid sequence of the AGA2 SP is as shown in SEQ ID NO:39, and the amino acid sequence of the SPP SP is as shown in SEQ ID NO:

45.

43. The cell according to any one of claims 28 to 42, wherein The complex is an MHC class I molecule or an MHC class II molecule, and preferably the complex is an HLA class I molecule or an HLA class II molecule.

44. The cell according to claim 43, wherein The α chain is encoded by HLA-DRA*01 or other alleles of the HLA-DRA family, and the β chain is encoded by HLA-DRB1*01 or HLA-DRB1*03 or HLA-DRB1*04 or HLA-DRB1*15 or other alleles of the HLA-DRB family; or The α chain is encoded by HLA-DQA1*01 or HLA-DQA1*03 or HLA-DQA1*05 or other alleles of the HLA-DQA1 family, and the β chain is encoded by HLA-DQB1*02 or HLA-DQB1*03 or HLA-DQB1*05 or HLA-DQB1*06 or other alleles of the HLA-DQB1 family; or The α chain is encoded by HLA-DPA1*01:03 or HLA-DPA1*02:02 or other alleles of the HLA-DPA1 family, and the β chain is encoded by HLA-DPB1*01:01 or HLA-DPB1*02:01 or HLA-DPB1*04:01 or HLA-DPB1*04:02 or other alleles of the HLA-DPB1 family.

45. A nucleic acid encoding a major histocompatibility complex (MHC), wherein: The complex comprises an α chain and a β chain, wherein the α chain is connected to a first protein binding domain, and the β chain is connected to a second protein binding domain, wherein the first protein binding domain and the second protein binding domain are capable of binding to form the complex, and the first protein binding domain and the second protein binding domain form an FcAB dimer to enhance the binding of the α chain and the β chain to form the MHC.

46. ​​The nucleic acid according to claim 45, wherein The encoded major histocompatibility complex is the major histocompatibility complex (MHC) according to any one of claims 1 to 17.

47. A method for preparing a major histocompatibility complex (MHC), wherein: The method comprises: Transforming a cell with the nucleic acid according to claim 45 or 46, And culturing the cell under conditions where the complex according to any one of claims 28 to 44 is expressed.

48. The method of claim 47, wherein: The cell is a yeast cell.

49. A method for identifying a peptide that binds to a major histocompatibility complex (MHC), wherein: The method comprises: i) contacting the cell according to any one of claims 28 to 44 with a peptide, and ii) detecting the binding of the peptide to the complex according to any one of claims 28 to 44, thereby identifying the peptide binding to the complex.

50. The method of claim 49, wherein: The peptides comprise mixtures of peptides.

51. The method of claim 49 or 50, wherein: The peptide competes with a reference peptide for binding to the complex.

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