Engineered cells for discovery of MHC neoantigen peptide epitopes

Abstract

The invention relates to an engineered cell capable of displaying a trimer of a major histocompatibility complex and an antigen peptide, a nucleic acid encoding the trimer, and application and a preparation method of the engineered cell. The engineered cells can be used for accurate and efficient high-throughput screening of MHC-II target peptides and development of new antigens and related vaccines and immunotherapies thereof.

Description

Technical Field

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

[0002] T cell-mediated immune responses play an important role in many diseases, including tumors, autoimmunity, transplant rejection, and infectious diseases. As the central regulator of adaptive immunity, the activation of CD4+T cells requires the specific recognition of antigens or pathogenic peptides bound and presented by major histocompatibility complex class II (MHC-II) through their receptors (TCR). MHC-II is a heterodimeric transmembrane protein composed of α and β chains, each chain containing two domains. These proteins capture antigenic peptides processed inside professional antigen presenting cells (APCs) and present them on the surface of APCs for CD4+T cells to recognize and initiate downstream immune responses. The peptide binding groove of MHC-II formed by the α1 and β1 domains contains several pocket-like spatial structures, which like to accommodate specific side chains of the "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 various peptide registers is crucial for the discovery of new tumor antigens, autoimmune regulation, vaccine design, prevention of infectious diseases, and alleviation of transplant rejection.

[0003] The human MHC, or human leukocyte antigen (HLA), is a highly polymorphic sugar-modified transmembrane protein known in nature. This polymorphism is associated with the presence of multiple naturally occurring HLA haplotypes, gene subtypes (DR, DQ, DP), and thousands of alleles. Moreover, the MHC proteins translated and expressed by each allele can bind and present multiple peptide fragments. Therefore, studying the peptide binding properties of various MHC-IIs and determining the epitope maps of various peptide antigens are extremely challenging problems, requiring high-throughput and high-content gene protein engineering methods. Among the conventional methods, some soluble recombinant MHC-II molecules are purified from different expression systems (such as B cell lines, insect cells, yeast, or E. coli), and then the binding strength of these molecules with different peptides is analyzed; other studies focus on evaluating the binding of peptides to MHC-II expressed on the surface of APCs. The peptide fragments used in these methods are either solid-phase synthesized by chemical methods or produced by genetic methods such as phage display. This inefficient synthesis of peptide fragments, labor-intensive preparation of soluble proteins, lengthy binding assays, or the generation of non-quantitative data (e.g., peptide abundance) limits 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 with the advantages of directed evolution can be used to express specified MHC-II alleles for 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, and therefore, it is a preferred platform for the development of this approach. Summary of the invention

[0004] The present application provides an engineered cell that can display a trimer of a major histocompatibility complex and an antigenic peptide, a nucleic acid encoding the trimer, and the use of the engineered cell and a method for preparing the engineered cell. The engineered cell can be used for accurate and efficient high-throughput screening of MHC-II target peptides.

[0005] Specifically, the present application relates to: 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:

[0006] The α chain is linked to a first protein binding domain to form a first fusion protein,

[0007] The beta chain is linked to a second protein binding domain to form a second fusion protein,

[0008] 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,

[0009] 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.

[0010] Furthermore, 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.

[0011] Furthermore, the cell is a yeast cell.

[0012] Furthermore, the single-chain domain is covalently fused to the molecule on the cell surface and is bound to the cell surface.

[0013] Furthermore, 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.

[0014] Further, the molecule is a protein.

[0015] Furthermore, the protein is endogenous to the cell.

[0016] Furthermore, the protein is Aga2p, a-lectin, α-lectin, flocculin, Cwp1p, Cwp2p or Tip1p.

[0017] Furthermore, the protein is Aga2p.

[0018] Furthermore, the amino acid sequence of Aga2p is shown in SEQ ID NO:1.

[0019] Furthermore, 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 registered 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 registered peptide or its variant is selected from the amino acid sequence shown in any one of SEQ ID NO: 20-38.

[0020] Furthermore, the amino acid sequence of the single-chain domain is shown in any one of SEQ ID NOs: 2-6 or 46-77.

[0021] Further, 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

[0022] 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.

[0023] Furthermore, 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;

[0024] 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.

[0025] Further, 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;

[0026] Preferably, the Fc A domain and FcB domain are capable of increasing the expression display amount and / or copy number of the complex;

[0027] Preferably, the FcA domain comprises only the first CH3 domain, and the FcB domain comprises only the second CH3 domain; or

[0028] 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;

[0029] 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.

[0030] Further, either or both of the FcA domain and the FcB domain comprise one or more amino acid modifications, and the modifications can achieve any one, two or three of the following:

[0031] (1) enhancing or stabilizing the FcA domain and / or the FcB domain itself ("FcS"),

[0032] (2) enhancing or stabilizing the covalent or non-covalent binding between the FcA domain and the FcB domain ("FcM"),

[0033] (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").

[0034] Furthermore, 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 SPPSP is as shown in SEQ ID NO:45.

[0035] Furthermore, 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.

[0036] Further, 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

[0037] 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

[0038] 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.

[0039] 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 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.

[0040] Furthermore, the major histocompatibility complex encoded by it is the aforementioned major histocompatibility complex (MHC).

[0041] A method for preparing a major histocompatibility complex (MHC), the method comprising:

[0042] Transforming cells with the aforementioned nucleic acid and nucleic acid encoding the single-chain domain,

[0043] and culturing the cells under conditions for expressing the aforementioned complex.

[0044] Furthermore, the cell is a yeast cell.

[0045] A method for identifying a peptide that binds to a major histocompatibility complex (MHC), the method comprising:

[0046] i) causing the aforementioned cell to display the single-chain domain library,

[0047] and ii) detecting the trimer on the surface of a monoclonal cell in the cell library displaying the aforementioned single chain domain, thereby identifying the binding peptide of the aforementioned complex.

[0048] Furthermore, 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.

[0049] Furthermore, the peptide, or a mutant of the peptide, or a library of mutants of the peptide, or a mixture of peptides non-covalently binds to the complex.

[0050] A registered peptide or a variant thereof capable of binding to a major histocompatibility complex (MHC), wherein the registered peptide or a variant thereof is selected from the amino acid sequence shown in any one of SEQ ID NOs: 20-38. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1CODAAH system design scheme. The peptide fused to the 2nd subunit of a-lectin protein (Aga2p) is fused to the N-terminus and C-terminus with HA and V5 epitope tags, respectively. The expression and cell surface display level of the peptide can be detected by immunofluorescence staining with specific antibodies to any tag (such as Anti-HA antibody intercalation). The extracellular domains of MHC-IIα and MHC-IIβ chains with C-terminal dimerization modifications are expressed under the guidance of another subtyping vector independent of the peptide display vector. The peptide is fused with the yeast secretory assembled a-lectin protein (formed by Aga1p and Aga2p subunits through two -SH to form -SS-) and anchored on the cell surface through the classic yeast display method. The MHC-IIα / β heterodimer improves the recombination efficiency through the enhanced dimerization of the C-terminus, and is anchored on the cell surface through non-covalent binding with specific antigenic peptides, realizing the peptide / MHC-IIα / β trimer yeast co-display. The relative fluorescence levels of different fluorophores coupled to anti-tag and anti-MHC-II (Anti-MHC-II) reagents indicate the degree of saturation of bound MHC-II pairs with specific binding peptides. EBY00: parental yeast strain for yeast display, GPI: glycosylphosphatidylinositol transporting yeast endogenous proteins, which are translated and expressed by the yeast genome.

[0052] Figure 2 A schematic diagram of the target gene region of the selectable plasmid is shown, and the target gene is the DR1 allele (HLA-DRA*01:01 / DRB1*01:01). Using the selectable plasmid, yeast can translate and express the extracellular domain of the MHC-II complex. The C-terminus of MHC-II is modified by a leucine fragment (LZA or LZB) to enhance dimerization coupling. Each expression cassette includes a GA1 or GAL10 promoter in the direction shown, a fusion protein with a C-terminal modified chain of HLA-II, and an MFα terminator.

[0053] Figure 3 and Figure 2 Similarly, a schematic diagram of the target gene region of the selectable plasmid is shown, wherein the target gene is the DR4 allele (HLA-DRA*01:01 / DRB1*04:01).

[0054] Figure 4 and Figure 2 Similarly, a schematic diagram of the target gene region of the selectable plasmid is shown, wherein the target gene is the DQ6 allele (HLA-DQA1*01:02 / DQB1*06:02).

[0055] Figure 5 and Figure 2Similarly, a schematic diagram of the target gene region of the selectable plasmid is shown, 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.

[0056] Figure 6 and Figure 5 Similarly, a schematic diagram of the target gene region of the selectable plasmid is shown, wherein the target gene is the DR4 allele (HLA-DRA*01:01 / DRB1*04:01).

[0057] Figure 7 and Figure 5 Similarly, a schematic diagram of the target gene region of the selectable plasmid is shown, wherein the target gene is the DQ6 allele (HLA-DQA1*01:02 / DQB1*06:02).

[0058] Figure 8 Immunofluorescence labeling and flow cytometry of surface DR or DQ protein conformation were performed for four yeast strains, including those that secrete only HLA-II (background control), display LZ modification (HLA-II-LZ), display FcAB modification (HLA-II-Fc), and display FcAB modification (HLA-II-Fc with AGA2 SP signal peptide for enhanced extracellular delivery (w / AGA2 SP). The flow cytometry results are displayed in histogram format. The median fluorescence intensity (MFI) of HLA-IIα / β alleles and DR or DQ proteins under each condition is shown in the figure. The level of HLA-II-LZ or HLA-II-Fc yeast display can be quantitatively analyzed by signal-to-noise difference (ΔMFI=MFI_LZ-MFI_BC or MFI_Fc-MFI_BC).

[0059] Fig. 9 For cells that only secrete DR1 (nopep+DR background control), only display DR-specific peptides (+HA 308-316 no DR control), showing HA 308-316 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 Peptide secretes LZ to modify DR4-LZ, which displays HA 308-316The peptide also secreted Fc-modified DR4-LZ with AGA2 SP signal peptide to enhance extracellular delivery. Six types of yeast strains were subjected to immunofluorescence co-labeling and flow cytometry for surface HA tag and DR protein conformation. The flow cytometry results were displayed in dot plots (SSC scatter vs HA tag) or histograms. Among them, the dot plots marked the percentage of gated HA+ positive cell subpopulations, and the histograms marked the median fluorescence intensity (MFI) of HA+ subpopulation DRα / β protein. DR display level can be as follows Figure 8 Quantitative analysis method.

[0060] Fig.10 Both display HA 308-316 The parent yeast library (+pep) in which the peptide variants secrete DR1 was subjected to immunofluorescence co-labeling and flow cytometry sorting for surface HA tag and DR protein conformation. The HA+ / DR+ double positives of the four rounds of sorting experiments (S1, S2, S3, S4) and the background control used in each round, i.e., the HA- / DR- double negatives that only secrete DR1 (no pep), are shown in the HA tag vs. DR protein dot plot. In addition, the SSC side scatter vs. HA tag dot plot is annotated with the percentage of gated HA+ positive cell subpopulations, and the histogram is annotated with the median fluorescence intensity (MFI) of the HA+ subpopulation DRα / β protein. The DR display level that increases successively in each round can be shown as follows. Figure 8 Quantitative analysis method.

[0061] Fig.11 Both display HA 308-316 Ten single clones selected from the yeast sub-library in which peptide variants secrete DR1 were subjected to yeast PCR and target peptide base sequencing. The results were compared with the DNA primers used to construct the library and the wild-type gene and its peptide amino acid sequence. Except for #1 and #4, the sequencing peak graph had obvious overlapping peaks, indicating that a single yeast can accommodate multiple recombinant plasmids formed by transformation. Therefore, the dominant codon DNA of clone #5 and clone #7 peptide variants is the same as the product corresponding to the dominant codon, and it cannot be confirmed that the peptide variant display recombinant plasmids obtained by transformation of these two yeast clones are exactly the same.

[0062] Fig.12 The selected four monoclonal clones (#1, #7, #9, #10) were subjected to yeast plasmid extraction, E. coli transformation, and subsequent extraction of one to three E. coli plasmids corresponding to each yeast and target peptide base sequencing. The results were compared with the wild-type gene and its peptide amino acid sequence. The selected peptide display plasmid was further transformed into yeast to construct a display HA 308-316The peptide variants were used to secrete DR1 strains and immunofluorescence co-labeling and flow cytometry of surface HA tags and DR protein conformations were performed. The flow cytometry results were displayed as dot plots (SSC scatter vs HA tag) or histograms. The dot plots were annotated with the percentage of gated HA+ positive cell subpopulations, and the histograms were annotated with the median fluorescence intensity (MFI) of HA+ subpopulation DRα / β proteins. The DR display level can be as follows Figure 8 Quantitative analysis method.

[0063] Fig.13 The display plasmid corresponding to the peptide numbered #1 was used to further transform yeast to construct a strain that not only displays the #1 peptide but also secretes DR4, and the surface HA tag and DR protein conformation were co-labeled by immunofluorescence and flow cytometry. The flow cytometry results are displayed in dot plots (SSC scatter vs HA tag) or histograms. 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 expressed as Figure 8 Quantitative analysis method. Specific implementation plan

[0064] The present application provides a scheme for making yeast co-express an antigen peptide gene and an MHC-II allele, and co-displaying the expressed protein product, i.e., peptide / MHC-II trimer, on the yeast surface. This scheme not only achieves the co-expression of complex MHC-II alleles in yeast, but also solves the problem of low co-display of peptide / MHC-II trimer. It greatly improves the efficiency of the research and 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.

[0065] Specifically, the scheme of the present application uses a yeast co-display system ( Figure 1 ), here named CODAAH (CO-Display of Antigen-ligand and Associated HLA-II). In order to make the peptide fragments and MHC-II complexes co-displayed on the cell surface in the form of trimers, the present application first connects the protein products to the yeast surface by genetically fusing the peptide fragment genes. Since the peptide fragments come from T cell antigens, the MHC-II complexes secreted in a soluble form can non-covalently bind to the peptide fragments displayed by yeast, thereby realizing the co-display of peptide / MHC-II trimers 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 has been significantly improved.

[0066] definition

[0067] 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 only as examples. Without departing from the present invention, those skilled in the art will now appreciate that many variations, changes and replacements are possible. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in the implementation of the present invention.

[0068] As used herein, the percentage of "identity", such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to the degree of similarity between amino acid sequences or between nucleotide sequences determined by sequence alignment, which is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%. For example, the proportion of the number of positions with the same base or amino acid residue to the total number of positions determined after two sequences have the same residue at as many positions as possible by introducing spaces, etc. The percentage of "identity" can be determined using software programs known in the art. Preferably, the comparison is performed using default parameters. A preferred comparison program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0069] 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). "Complete complementarity" refers to the formation of hydrogen bonds between all consecutive residues of a nucleic acid sequence and 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% in 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 a single base or a single nucleotide, according to the Watson-Crick base pairing principle, when A is paired with T or U, C is paired with G or I, it is called complementary or matching, and vice versa; and other base pairings are called 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.

[0070] 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% as the best, and have the same or substantially similar properties or functions compared to the native or parent protein or enzyme.

[0071] In this article, "coding" refers to i) genetic information contained in a DNA sequence that can be transcribed into an RNA molecule, and / or ii) genetic information contained in an 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 that can be translated into a protein in an mRNA precursor or mature mRNA, and can also refer to a complementary sequence or a fragment thereof of a deoxyribonucleotide (DNA) sequence that is used as a template to transcribe the mRNA precursor or mature mRNA. In addition, the "coding sequence" of the present application may further include polynucleotide sequences encoding 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.

[0072] The term "MHC-II" or "MHC-II complex" as used herein 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, to regulate the immune system by presenting antigenic peptides. The MHC-II complex includes two human leukocyte antigen (HLA) proteins, referred to herein as "alpha chains" or "α chains" and "beta chains" or "β chains," which combine to form heterodimers. The α1 and β1 regions of the α and β chains are respectively close to and aggregated 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 an immunoglobulin-like domain. 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 via a transmembrane domain.

[0073] 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, thereby providing 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 from antigens outside the cell. MHC-II class proteins include HLA-DP, HLA-DQ and HLA-DR, heterodimeric cell surface receptors including alpha and beta chains.

[0074] The terms "HLA-DR4 α chain" or "HLA-DR4 α chain protein" provided herein include any recombinant or naturally occurring form of the human leukocyte antigen (HLA) HLA-DR4 α chain protein, also known as MHC-II class DRA, or variants or homologs thereof that retain HLA-DR4 α chain protein activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HLA-DR4 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 consecutive amino acid portion) compared to a naturally occurring HLA-DR4 α chain polypeptide. In an embodiment, the HLA-DR4 alpha chain is the protein identified by UniProt sequence reference P01903, a homologue or a functional fragment thereof.

[0075] The terms "HLA-DR β chain" or "HLA-DR β chain protein" provided herein include any recombinant or naturally occurring form of 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 the activity of HLA-DR1 or DR4 or DR15 or other DR β chain proteins (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.

[0076] The terms "HLA-DQ6 α chain" or "HLA-DQ6 α chain protein" provided herein include any recombinant or naturally occurring form of the human leukocyte antigen (HLA) DQ α1 chain (HLA-DQ6 α chain), also referred to as DC-1 α chain, DC-α, HLA-DCA, MHC-II class 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% 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.

[0077] The terms "HLA-DQ6 beta chain" or "HLA-DQ6 beta chain protein" provided herein include any recombinant or naturally occurring form of human leukocyte antigen (HLA) DRB1 beta chain 1 protein (HLA-DQ6 beta chain), also known as MHC-II class 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 consecutive 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.

[0078] 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., 50, 100, 150, or 200 consecutive amino acid portions) 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.

[0079] "Contacting" is used in its simple common sense and refers to the process of allowing at least two different substances (e.g., a peptide and an MHC-II complex) to become close enough 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, a nucleic acid and a cell as provided herein. In embodiments, contacting includes, for example, allowing a nucleic acid as described herein to enter a cell.

[0080] As used herein, "cell" refers to a cell that performs a metabolic or other function 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., fall armyworms), and human cells.

[0081] The term "recombinant" when referring to, for example, a cell, nucleic acid, protein or vector, 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. Nucleic acid molecules 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 cells in their natural (non-recombinant) form, or express native genes that are abnormally expressed, under-expressed or not expressed at all. Transgenic cells and plants are those cells and plants that express heterologous genes or coding sequences, usually as a result of recombinant methods.

[0082] The term "heterologous" when used to refer to portions of a nucleic acid means 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 typically 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 means 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).

[0083] 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 a 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.

[0084] 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.).

[0085] "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.

[0086] "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 (undiseased) individual (e.g., a standard control subject). A standard control can also represent an average measurement or value collected from a group of healthy individuals without a given disease (i.e., a standard control population), such as 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, controls 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 importance 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. Among them, those skilled in the art will recognize that standard controls can be designed to evaluate any number of parameters (e.g., RNA levels, protein levels, specific cell types, specific body fluids, specific tissues, etc.).

[0087] 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.

[0088] 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 to which the present application belongs. Therefore, those of ordinary skill in the art should recognize that the scope of the present application also includes the improvements and modifications to the aspects and embodiments.

[0089] cell

[0090] 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:

[0091] The α chain is linked to a first protein binding domain to form a first fusion protein,

[0092] The beta chain is linked to a second protein binding domain to form a second fusion protein,

[0093] 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 prompt 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.

[0094] 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 a brewer's yeast. In some embodiments, the yeast belongs to strain EBY100 (Boder & Wittrup, Yeast surface display for screening combinatorial polypeptide libraries, 1997).

[0095] 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 be directly or indirectly bound to the cell wall glucan or cell wall mannan. In some embodiments, the structure that can anchor the yeast cell wall can be directly or indirectly covalently or non-covalently bound to the cell wall glucan or the cell wall mannan. In some embodiments, the structure that can anchor the yeast cell wall can be directly or indirectly covalently bound to the cell wall glucan or non-covalently bound to the cell wall mannan. In some embodiments, the structure that can anchor the yeast cell wall and / or the secretion signal peptide are derived from the α-condensin system and / or the flocculin system (glycosylphosphatidylinositol anchor system and flocculin domain anchor system). In some embodiments, the structure that can anchor the yeast cell wall is directly or indirectly combined or connected to the GPI anchor attachment signal region or the flocculation functional region. In some embodiments, the molecule is endogenous to the cell. In some embodiments, the molecule is selected from: Aga2p, a-lectin, α-lectin, flocculin, Cwp1p, Cwp2p or Tip1p. In some embodiments, the protein is Aga2p. In some embodiments, Aga2p is a protein encoded by a gene with a GENE ID of 852851 in the NCBI database. Aga2p, i.e., Aga2 protein, is a binding subunit of α-condensin, which can bind to the α-condensin core subunit Aga1 protein through a disulfide bond. In some embodiments, Aga2p is a protein encoded by a gene with a GENE ID of 852851 in the NCBI database. In some embodiments, Aga2p has 1, 2, 3, 4, 5, 6 or more mutations in its amino acid sequence compared to the protein encoded by the gene with a GENE ID of 852851 in the NCBI database. In some embodiments, Aga2p does not have an S288C mutation in its amino acid sequence compared to the protein encoded by the gene with a GENE ID of 852851 in the NCBI database.In some embodiments, the amino acid sequence of Aga2p comprises the amino acid sequence as shown in SEQ ID NO: 1, or a conservatively substituted variant of the amino acid sequence as shown 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 with the amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of Aga2p is as shown in SEQ ID NO: 1.

[0096] 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 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 by it 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 the present application, and, 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.

[0097] In some embodiments, the amino acid sequence of the registered peptide or its mutant is shown in SEQ ID NO: 20-38.

[0098] 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 homologous or heterologous dimers (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. WO2009 / 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 method, the first protein binding domain and the second protein binding domain may contain mutations for the corresponding binding structure of the above method. In some embodiments, the first protein binding domain and the second protein binding domain are bound to each other via a disulfide bond.

[0099] 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.

[0100] 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 Jun Zipper and the second leucine zipper domain is c-Fos leucine zipper domain (Fos Zipper). In some embodiments, the acidic leucine domain comprises an amino acid sequence as set forth in SEQ ID NO: 7, or a conservatively substituted variant of the amino acid sequence as set forth in 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 as set forth in SEQ ID NO: 7. In some embodiments, the amino acid sequence of the acidic leucine domain is an amino acid sequence as set forth in SEQ ID NO: 7, or a conservatively substituted variant of the amino acid sequence as set forth in 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 as set forth in SEQ ID NO: 7.

[0101] In some embodiments, the basic leucine domain comprises an amino acid sequence as set forth in SEQ ID NO: 8, or a conservatively substituted variant of the amino acid sequence as set forth in 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 with the amino acid sequence as set forth in SEQ ID NO: 8. In some embodiments, the amino acid sequence of the basic leucine domain is an amino acid sequence as set forth in SEQ ID NO: 8, or a conservatively substituted variant of the amino acid sequence as set forth in 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 with the amino acid sequence as set forth in SEQ ID NO: 8.

[0102] In some embodiments, the Fos Zipper comprises an 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 with the amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the amino acid sequence of the Fos Zipper is an 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 with the amino acid sequence as set forth in SEQ ID NO: 9.

[0103] In some embodiments, the Jun Zipper comprises an amino acid sequence as set forth in SEQ ID NO: 10, or a conservatively substituted variant of the amino acid sequence as 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 with the amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, the amino acid sequence of the Jun Zipper is an amino acid sequence as set forth in SEQ ID NO: 10, or a conservatively substituted variant of the amino acid sequence as 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 with the amino acid sequence as set forth in SEQ ID NO: 10.

[0104] 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;

[0105] 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.

[0106] In some embodiments, the first protein binding domain and the second protein binding domain both comprise the Fc domain of an immunoglobulin. After being digested by papain, the 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 the present application, the term "Fc domain" is a monomer, which may refer to any portion other than the CH1 segment in the constant region of any heavy chain of an immunoglobulin. In some embodiments, the FcA and FcB may be derived from the Fc segment of the same immunoglobulin, or from the Fc segment of different immunoglobulins. In some embodiments, the immunoglobulins from which the FcA and FcB are derived may be selected from: IgG1, IgG, IgE, IgM, IgD and IgA. In some embodiments, the immunoglobulins from which the FcA and FcB are derived may be selected from: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. In some embodiments, the FcA and FcB are selected from the group consisting of amino acid sequences as set forth in SEQ ID NOs: 11-19, conservative substitution variants of the amino acid sequences as 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 with the amino acid sequences as set forth in SEQ ID NOs: 11-19. For example, in some embodiments, the amino acid sequences of the FcA and FcB are both as set forth in SEQ ID NO: 11, or conservative substitution variants of the amino acid sequences as set forth in SEQ ID NO: 11, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences as set forth in SEQ ID NOs: 11. In some embodiments, the amino acid sequence of FcA is as shown in SEQ ID NO:12, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:12, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:12; and the amino acid sequence of FcB is as shown in SEQ ID NO:13, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:13, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:13.In some embodiments, the amino acid sequence of the FcA is as shown in SEQ ID NO:4, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:13, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:13; and the amino acid sequence of the FcB is as shown in SEQ ID NO:12, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:12, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:3. In some embodiments, the amino acid sequence of FcA is as shown in SEQ ID NO:14, or is a conservatively substituted variant of the amino acid sequence as shown in SEQ ID NO:14, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:14; and the amino acid sequence of FcB is as shown in SEQ ID NO:15, or is a conservatively substituted variant of the amino acid sequence as shown in SEQ ID NO:15, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:15. In some embodiments, the amino acid sequence of FcA is as shown in SEQ ID NO:15, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:15, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:15; and the amino acid sequence of FcB is as shown in SEQ ID NO:14, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:14, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:14.In some embodiments, the amino acid sequence of FcA is as shown in SEQ ID NO:16, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:16, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:16; and the amino acid sequence of FcB is as shown in SEQ ID NO:17, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:17, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:17. In some embodiments, the amino acid sequence of FcA is as shown in SEQ ID NO:17, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:17, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:17; and the amino acid sequence of FcB is as shown in SEQ ID NO:16, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:16, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:16. In some embodiments, the amino acid sequence of FcA is as shown in SEQ ID NO:18, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:18, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:18; and the amino acid sequence of FcB is as shown in SEQ ID NO:19, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:19, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:19.In some embodiments, the amino acid sequence of FcA is as shown in SEQ ID NO:19, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:19, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:19; and the amino acid sequence of FcB is as shown in SEQ ID NO:18, or is a conservative substitution variant of the amino acid sequence as shown in SEQ ID NO:18, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:18.

[0107] In some embodiments, the FcA domain and the FcB domain can increase the expression display amount and / or copy number of the complex. In some embodiments, the FcA domain only includes the first CH3 domain, and the FcB domain only includes the 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 includes both the first CH2 domain and the first CH3 domain, and the FcB domain includes both the second CH2 domain and the second CH3 domain; wherein the first CH3 domain and the second CH3 domain may be from the same immunoglobulin subtype or different immunoglobulin subtypes; the first CH2 domain and the second CH2 domain may be from the same immunoglobulin subtype or different immunoglobulin subtypes; the first CH2 domain and the first CH3 domain may be from the same immunoglobulin subtype or different immunoglobulin subtypes, and the second CH2 domain and the second CH3 domain may be from the same immunoglobulin subtype or different immunoglobulin subtypes. Depending on the different immunoglobulins from which they are derived, for example, when the FcA and / or FcB are from IgE or IgM, in some embodiments, the FcA domain may further include a first CH4 domain, and the FcB domain may further include a second CH4 domain.

[0108] In some embodiments, the first CH2 domain and the first CH3 domain of the first protein binding domain are directly or indirectly connected through 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 through a linker. In some embodiments, the first CH2 domain and the first CH3 domain of the first protein binding domain are indirectly connected through a linker; and the second CH2 domain and the second CH3 domain of the second protein binding domain are indirectly connected through 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, and the connecting peptide 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 to have different lengths or functions. The types, functions and characteristics of the connecting peptide can be referenced to, 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.

[0109] In some embodiments, either or both of the FcA domain and the FcB domain comprise one or more amino acid modifications that can (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 caused by mispairing of the FcA domain and the FcB domain ("FcN").

[0110] 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.

[0111] Preferably, the signal peptide for initiating the 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 as shown in SEQ ID NO:39, and the amino acid sequence of SPP SP is as shown in SEQ ID NO:45.

[0112] 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.

[0113] 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

[0114] 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

[0115] 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.

[0116] Engineered Nucleic Acid Molecules

[0117] 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. Wherein 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. Wherein the MHC is the MHC contained in the engineered cell described in the first aspect of the present application.

[0118] 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, the DNA molecule, in addition to comprising a nucleic acid fragment encoding the MHC or the trimer, also comprises a gene manipulation or regulatory element for replication and / or expression in a prokaryotic and / or eukaryotic cell.

[0119] The necessary structural elements that make the engineered DNA molecule replicate or efficiently replicate in cells are known in the art, including, for example, an origin of replication (ORI). In some embodiments, the engineered DNA molecule further comprises a marker gene or its fragment and / or a reporter gene or its fragment and a unique restriction endonuclease site that allows insertion of DNA elements, preferably a restriction endonuclease site in the form of a multiple cloning site (MCS). The marker gene is conducive to identifying cells containing a plasmid comprising the marker gene, and can be selected from, for example, an antibiotic resistance gene. Each restriction endonuclease site in the MCS can be specifically recognized by different restriction endonucleases.

[0120] 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, and linear molecules that can be replicated in prokaryotes. Plasmids can replicate, i.e., amplify in cells independently of the genomic genetic information stored in the nucleoid or nucleoid of prokaryotes, 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 such vectors: they are based on pUC, pTZ plasmids or any other plasmids (e.g., pMB1, pCoIE1) etc. that contain an ORI that supports high copies of plasmids.

[0121] In some embodiments, the engineered DNA molecule is a DNA molecule or a fragment thereof constituting a prokaryotic nucleoid or nucleoid, or a DNA molecule or a fragment thereof constituting a eukaryotic genome, that is, the coding sequence or its complementary sequence comprising the aforementioned MHC or trimer can be replicated along with the prokaryotic genome.

[0122] In some embodiments, the engineered DNA molecule can be transcribed into mRNA. In some embodiments, the engineered DNA molecule also comprises a coding sequence of an element that can be used to start or regulate the expression of the protein, polypeptide or its fragment after transcription, and the element includes but is not limited to 5'UTR, 3'UTR, poly (A) tail (or tailing signal), etc. In some embodiments, the engineered DNA molecule comprises a coding sequence of at least one untranslated region (UTR). In some embodiments, the engineered DNA molecule comprises at least a coding sequence of 5'UTR and a coding sequence of the protein, polypeptide or its fragment. In some embodiments, the engineered DNA molecule comprises at least a coding sequence of 5'UTR, a coding sequence of the MHC or the trimer, a coding sequence of 3'UTR, a tailing signal (or a DNA sequence corresponding to the Ploy (A) tail sequence) from 5' to 3', and the start codon (5' end) and the stop codon (3' end) may also be included at both ends of the coding sequence of the MHC or the trimer, which are the first three nucleotides and the last three nucleotides that can be translated of the mRNA molecule, respectively. The 5'UTR usually contains 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. The RBS promotes the efficient and accurate translation of mRNA molecules by recruiting ribosomes at the start of translation. 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, the 5'UTR includes an internal ribosome entry site or IRES. The 3'UTR may contain one or more regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of mRNA molecules, binding sites for regulatory RNA molecules (such as miRNA molecules), and / or signal sequences that participate in the intracellular transport of mRNA molecules.

[0123] On the basis of the aforementioned embodiments, 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 the stability of mRNA molecules, binding sites for amino acid sequences that enhance the translation of mRNA molecules, 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, an out-of-frame upstream translation initiation site, an out-of-frame upstream start codon, and / or a nucleotide sequence that produces a secondary structure that reduces or prevents translation. The presence of such nucleotide sequences in the 5'UTR can have a negative impact on translation.

[0124] The coding sequence of the MHC or the trimer comprises codons that can be translated into an amino acid sequence. All the codons contained in the coding sequence may be naturally occurring codons encoding amino acids, or may be partially or entirely composed of 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 non-natural amino acids.

[0125] In some embodiments, the engineered DNA molecule further comprises a structural element necessary for initiating or regulating transcription of the RNA on the 5' end side of the target gene fragment, and the structural element is known in the art. In some embodiments, the structural element comprises at least a promoter. Promoters and their sequences are known in the art, including weak promoters, medium strength promoters, strong promoters, mini promoters or core promoters, etc. In some specific embodiments, the promoter is a strong promoter. In some embodiments, the promoter can initiate transcription of the coding sequence of the MHC or the trimer in prokaryotes. In some embodiments, the promoter can initiate transcription of the coding sequence of the MHC or the trimer in eukaryotic cells. The "promoter" comprises at least one transcription recognition site and a subsequent transcription factor binding site. The recognition and binding sites can interact with amino acid sequences that mediate or regulate transcription. Compared with the recognition site, the binding site is closer to the aforementioned target gene fragment. 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 of the enhancing promoter activity between the recognition site and the binding site. In some embodiments, the promoter is a strong promoter, i.e., the promoter comprises a sequence that promotes the transcription of the aforementioned RNA coding sequence. Strong promoters are 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 other regulatory elements before the promoter, such as an enhancer that can promote the transcription of the aforementioned RNA coding sequence in a DNA plasmid.

[0126] In some embodiments, the eukaryotic cell is a yeast cell. In some embodiments, the DNA molecule is a yeast display vector.

[0127] 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.

[0128] In some embodiments, the engineered nucleic acid molecule may also be a hybrid molecule of DNA and RNA.

[0129] In addition, the present application also covers any cells comprising the above-mentioned engineered nucleic acid molecules.

[0130] Preparation and identification methods

[0131] The present application also relates to a method for preparing a major histocompatibility complex (MHC) or a trimer formed by non-covalent bonding of MHC and a single-chain domain, the method comprising transforming a cell using the engineered nucleic acid molecule of the second aspect, and culturing the cell under conditions of expressing the complex or the 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 belongs to the yeast strain EBY100.

[0132] The present application also relates to a method for identifying a peptide that binds to a major histocompatibility complex (MHC), the method comprising:

[0133] i) allowing the cell of the first aspect to display the single chain domain library, and

[0134] ii) detecting the trimer on the surface of the monoclonal in the cell library displayed by the single-chain domain, thereby identifying the peptide bound 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 with 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.

[0135] Example

[0136] Example 1: Yeast cell co-display dependent on peptide binding and HLA-linked C-terminal domains

[0137] As a transmembrane protein, the stability of the heterodimerization of MHC-II α and β chains depends to a certain extent on the cooperation of the transmembrane domain and intracellular domain of each α and β chain. In order to use eukaryotic single-cell organisms such as yeast to express stable human MHC-II extracellular domains (excluding transmembrane domains and intracellular domains), the inventors first selected protein heterodimers corresponding to the most representative alleles of MHC-II human leukocyte antigens 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 a yeast shuttle vector that can express DR1, DR4, or DQ6 recombinant extracellular domain complexes. The bidirectional promoter GAL1-10 in the designed vector plasmid can guide the simultaneous expression of the α and β chains, and the C-termini of the expressed α and β chains are fused with a leucine zipper domain (LZA or LZB to form an LZ stable dimer, the sequences selected in the embodiment are shown in SEQ ID NO: 9 and SEQ ID NO: 10) or an antibody heavy chain constant domain (FcA or FcB to form an Fc stable dimer, the sequences selected in the embodiment are shown in SEQ ID NO: 15 and SEQ ID NO: 16) so that the HLA-II α / β chain with LZ or Fc modification is more likely to form a natural HLA complex ( Figure 2-7 At the same time, the synthetic SPP SP (as shown in SEQ ID NO: 45) signal peptide was further replaced by the yeast endogenous AGA2 SP (as shown in SEQ ID NO: 39) signal peptide at the N-terminal of the Fc-modified DRβ chain or DQα chain. Although both LZ and Fc fused to the C-terminus can promote the stable heterodimerization of HLAα and β chains, the results of flow cytometry showed that the expression level of HLA recombinant protein modified by Fc on the yeast surface was significantly higher than that of HLA recombinant protein modified by LZ ( Figure 8 ). This result confirms that C-terminal modification of the designed HLA fusion protein, especially with AGA2 SP signal peptide to guide extracellular delivery and / or Fc modification can effectively promote yeast expression of HLA extracellular domain.

[0138] 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 that yeast cells display peptide / MHC-II trimers. Among them, the expression of antigenic peptides and HLA-DRα / β heterodimers is driven by two sets of yeast shuttle vector plasmids. First, regardless of whether the peptide / MHC-II trimer is formed, since the antigenic peptide is expressed by the peptide display plasmid alone, the daughter yeast cells transformed with this type of plasmid can 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 ( Fig. 9 ). Among them, the vector for directing the expression of the single-chain fusion antigen peptide gene uses the traditional yeast display method (see, for example, Boder & Wittrup, Yeast surface display for screening combinatorial polypeptide libraries, 1997). In order to successfully form dimers of the various representative HLA allele proteins selected, and successfully display them on the yeast surface by non-covalent binding (rather than direct display) with the single-chain fusion antigen peptide, the inventors first knocked out the coding gene of the Aga2p protein used for yeast display of HLA in the aforementioned HLA display plasmid from the plasmid, and constructed an HLA secretion vector that only secretes but does not display the HLA dimer structure skeleton with C-terminal enhancement modification ( Figure 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 in the cell, ultimately anchoring the peptide / HLA trimer structure on the cell surface ( Figure 1 The vector for displaying single-chain fusion antigen peptide and the C-terminal enhanced modified HLA secretory vector were transformed into Saccharomyces cerevisiae EBY100 for protein expression and trimer co-display. Using double antibody fluorescent labeling and flow cytometry detection technology, the inventors successfully demonstrated that the CODAAH system can display peptide / HLA trimers on the cell surface ( Fig. 9 ). More importantly, the C-terminus of HLA was modified, especially the Fc modification, which greatly increased the expression abundance and display of peptide / HLA trimers formed by DR1 on the yeast surface (expressed as the mean or median of the fluorescence signal intensity (MFI), with the signal enhanced by more than 2 times). This method can enable some HLA allele proteins that are difficult to co-display on the yeast surface to successfully form peptide / HLA trimers (expressed as the mean or median of the fluorescence signal intensity, the signal increases from zero to one).

[0139] Example 2: Construction of a display peptide library to achieve CODAAH system optimization and antigen peptide development

[0140] The co-display of peptides / HLA trimers by the CODAAH system depends on the modification and stabilization of the HLA-II α / β heterodimer protein itself and on the preference for HLA-II specific antigenic 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. Using PCR primers carrying five degenerate codons (NNS) to amplify the insert fragment and double enzyme digestion to obtain a linearized empty plasmid, the inventors injected a high concentration of a vector carrying a coding HA 306-318 The inserted fragment of the peptide variant sequence (as shown in SEQ ID NO: 46) and the linearized empty plasmid were transformed into yeast cells that can secrete and express DR1. The inserted fragment and the linearized empty plasmid formed a plasmid displaying the peptide variant through homologous recombination in the progeny 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 (signals can be represented by HA tags) and DR (signals can be represented by L243 monoclonal antibody) to obtain the target yeast clone. After 4 rounds of enrichment and sorting, the progeny library (accounting for about 0.01% of the parental library, ~10 4 The HA+ / DR+ double positive signal of yeast clones was significantly improved compared with the double positive signal of the parental library ( Fig.10 ). The sequencing results of the plasmids contained in the sub-library single clones showed that 90% of the clones carried peptide variants with base mutations ( Fig.11). From the bacterial PCR sequencing results directly performed on yeast clones, most of the designed degenerate codon positions 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 completely the same at the five anchor residue positions. It is further inferred that the dominant codon DNA of the peptide variants of yeast clone #5 and clone #7 are the same as the products corresponding to the dominant codons, 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 single clone yeasts numbered #1, #7, #9, and #10 and transformed Escherichia coli. The results of plasmid extraction and sequencing from multiple selected Escherichia coli verified this conjecture: a single yeast cell can accommodate at least 3 peptide variant display plasmids, and each peptide variant expressed by the plasmid can bind to DR1( Fig.12 In addition, the yeast PCR sequencing showed no overlapping peaks in clone #1, which indeed contained only one peptide variant (the sequencing results of E. coli extracted plasmids #1-1 and #1-2 were exactly the same as the yeast #1 PCR sequencing results), while the clones #7, #9, and #10 that showed overlapping peaks also contained more than one peptide variant. This conclusion indicates 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 of 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 promote the improvement of DR1 affinity, thereby facilitating the formation of trimers of co-displayed peptides and DR1 in the CODAAH system.

[0141] The effect of constructing library-preferred peptide variants to promote peptide / HLA trimer co-display may even improve the application of the CODAAH system in other related peptide / DR co-displays. 306-318 compared to( Fig. 9 ), peptide variant #1 not only improved its ability to bind to DR1, but also improved its ability to bind to DR4 in DR4-secreting yeast transformant strains ( Fig.13 ). And importantly, 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 ( Fig.13 ), which once again verified the optimal effect of C-terminal Fc modification on stabilizing HLA-IIα / β heterodimers. 306-318Variant library discovers DR1 new antigen peptides. Similarly, constructing a #1 variant library with peptide variant #1 as the wild type can be used as a method for discovering DR4 new antigens. It is worth mentioning that by 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 new antigen 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 optimizing peptides and discovering HLA new antigen peptides.

[0142] Materials and Methods Used in Examples 1 and 2

[0143] Unless otherwise specified, all materials and reagents used can be obtained from commercial sources. Unless otherwise specified, all experimental methods used are conventional methods.

[0144] Construction and electroporation of yeast co-display shuttle vector

[0145] The entire gene expression cassette (GAL1-10 / / AGA2-HA / / scFv 4-4-20 / / MFαTerm.) was cut out from pCT302 using KpnI and SacI, and then subcloned into the yeast shuttle vector pRS315 partially digested with KpnI / SacI to form a yeast surface display plasmid with a LEU nutritional marker gene. Then, an oligonucleotide DNA chain encoding an antigenic peptide (e.g., influenza virus hemagglutinin residues HA306-318: SEQ ID NO: 2) and a short spacer (GGGS) and a V5 epitope tag was used to replace the DNA chain encoding scFv4-4-20 in the original plasmid to construct a vector for displaying Aga2-single-chain antigenic peptide fusion protein.

[0146] The vector 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 for yeast display of C-terminal LZ-enhanced HLA-DR4 and HLA-DQ6 extracellular domain heterodimers, respectively. In order to create a vector for yeast display of C-terminal LZ-enhanced HLA-DR1 extracellular domain heterodimers, the β gene HLA-DRB1*04:01 in ptDR4-LZ was replaced with HLA-DRB1*01:01. In order to create a vector for yeast secretion of C-terminal enhanced modified HLA extracellular domain heterodimers, the nucleic acid sequences encoding AGA2 and HA-tags downstream (3'-end) of the DRβ gene or DQ6α gene of the above three plasmids for yeast display of HLA-II were replaced with stop codons in the same reading frame. The two sets of vectors obtained in this way can be used to display or secrete three representative HLA fusion proteins, DR1, DR4 and DQ6, with enhanced modification of C-terminal leucine binding domain (refer to Figure 2-4 ). Further, by molecular cloning, the first leucine zipper fragment and the second leucine zipper fragment at the downstream (3'-end) of the HLAα and β genes in each of the two groups of three vectors (one group of three display types and the other group of three secretion types) were replaced with antibody crystallizable fragments. In this way, the obtained two new groups of three new vectors (one group of three display types and the other group of three secretion types) can respectively guide the expression of C-terminal crystallizable antibody constant domain enhanced modified DR1, DR4 and DQ6 fusion proteins (reference Figure 5-7 Then, the nucleic acid sequence encoding the signal peptide of AGA2SP (as shown in SEQ ID NO: 39) is cloned into the N-terminus of the Fc-modified DRβ chain or DQα chain to replace the original nucleic acid sequence encoding the signal peptide of SPP SP (as shown in SEQ ID NO: 45).

[0147] The TRP+ vector plasmids secreting or displaying HLA-II were transformed into the Saccharomyces cerevisiae strain EBY100 (GAL1-AGA1:URA3 ura3-52trp1 leu2Δ1his3Δ200pep4:HIS2 prb1Δ1.6R can1 GAL) together with the LEU+ vectors that can direct the expression of single-chain antigen peptide fusion proteins or each plasmid was transformed separately. Electroporation was performed using a MicroPulser electroporator (BioRad), and the basic method was referred to the BioRad MicroPulser manual. After culturing in a 30°C incubator for 2-3 days, the yeast cell monoclones that received the TRP+ and / or LEU+ vectors formed single yeast colonies visible to the naked eye on solid agar plates containing tryptophan and / or leucine nutrient deficiency. The culture medium SD-SCAA contained 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.

[0148] Preparation of co-display yeast for flow cytometry

[0149] Inoculate 2 ml of SD-SCAA medium with a single yeast colony and culture in a 30°C shaker until the colony reaches 2.5-5.0×10 7 To induce protein expression promoted by GAL1-10, cells were collected by centrifugation for 10 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, the induced cells were first washed 1-2 times with 400 μL Tris-buffered saline (137 mM NaCl, 20 mM Tris-Cl, pH 7.6), and 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 at 23°C for at least 48 hours 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). 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 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, then incubated on ice for 10 minutes. After centrifugation to remove the primary reagent, the cells were washed again with ice-cold PBS + 1% BSA and resuspended in 40 μL 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 then resuspended in 500-700 μL PBS + 1% BSA for flow cytometry. 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 were unchanged.

[0150] Construction of a peptide variant / HLA trimer co-display yeast library and single clone sorting and analysis

[0151] 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:

[0152] 5'-TGGTGGTGGTTCTGCTAGCCCTAAGNNSGTGAAGNNSAATNNSNNSAAGNNSGCAACCGGAGGTGGACCCGGGAAGCCTATAC-3'(P0)(SEQ ID NO.78)

[0153] 5'-AGGCTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGGTGGTGGTGGTTCTGCTAGC-3'

[0154] (P1) (SEQ ID NO.79)

[0155] 5'-TGGCATGCTTAAGTAGAATCGAGACCGAGGAGTGGATTTGGTATAGGCTTCCCGGGTCC-3'(P2)(SEQ ID NO.80)

[0156] Among them, P0 is used in 5 HA 306-318 Random mutations were introduced at the peptide anchor residue positions, and P1 and P2 were used to amplify gene inserts carrying peptide mutants and add homologous sequences to both ends of the linearized vector. The linearized peptide display vector was obtained by double digestion with XmaI / NheI. A total of 50 μg of inserts and linearized vectors (1:1 mass ratio) after centrifugal concentration and drying were electrotransformed into 800 μl of yeast competent cells that can secrete DR1 to obtain a yeast library for co-display. The immunofluorescence labeling of the yeast library was amplified in equal proportions based on the labeling method of the aforementioned yeast strain. The sorting of yeast cells co-labeled with HA tags and DR was completed on a FACSAriaIIu flow cytometer. The sorted sub-library was re-amplified in SD medium, and then induced for expression in SG medium for flow cytometric analysis or further sorting. The sub-library selected and amplified for the last time was sampled and coated with agarose medium for monoclonal yeast PCR and gene sequencing, and then further yeast plasmid extraction, E. coli transformation and subsequent plasmid extraction and gene sequencing analysis of multiple E. coli corresponding to one yeast. The sequencing-verified peptide variant display plasmid was re-transformed into yeast competent cells that can secrete DR1 or DR4 for trimer co-display verification. The verification method can refer to flow cytometry analysis and the following quantitative analysis method.

[0157] Flow cytometric detection and quantification of relative binding

[0158] 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). Peptides (e.g., influenza virus hemagglutinin peptide HA 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.

[0159] Wherein, MFI(HA-) and MFI(HA+) represent the mean fluorescence intensity of HA-tag-conjugated AlexaFluor 488 emission of negative and positive cell populations, respectively, calculated using FlowJo. Analysis of peptide display levels on the yeast surface using anti-V5-tag staining yielded cMFI values ​​comparable to those described above.

[0160] HLA display levels dependent on peptide binding can be correctly calculated by using the normalized, background-corrected fluorescence with anti-DR staining and peptide display levels (DR-ratio) (anti-DQ staining is calculated similarly)

[0161]

[0162] Among them, (+) and (-) represent the yeast with co-display and the yeast with peptide only, respectively. MFI(DR) and cMFI represent the mean fluorescence intensity of DR-conjugated AlexaFluor 647 and HA 306-318 Display levels on the surface of the corresponding yeast strains. Normalization minimizes the error between experiments due to laser power output, detector amplification, and other cytometer parameters.

[0163] 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 only exemplary sequences of the embodiments of the present application, and are not any limitation to the embodiments of the present application.

[0164] Sequence Listing

[0165]

[0166]

[0167]

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.

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