Peptide-mhc ii protein constructs and uses thereof

Abstract

Provided herein are compositions comprising an MHC ligand peptide covalently attached to an MHC class II molecule. In some compositions, the MHC ligand peptide is covalently attached to the MHC class II molecule by a peptide linker, wherein the MHC ligand peptide or the peptide linker comprises a first cysteine, wherein the MHC class II alpha chain or portion thereof or the MHC class II beta chain or portion thereof comprises a second cysteine, and wherein the first cysteine and the second cysteine form a disulfide bond such that the MHC ligand peptide is bound in the peptide binding groove formed by the MHC class II alpha chain or portion thereof and the MHC class II beta chain or portion thereof. Also provided are nucleic acids encoding such compositions and methods of using such compositions to elicit an immune response in a subject.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Application No. 62 / 942,344, filed December 2, 2019, which is incorporated herein by reference in its entirety for all purposes.

[0003] References to sequence lists

[0004] Submit as a text file via EFS WEB

[0005] The sequence list written to file 696193SEQLIST.txt is 50.9 kilobytes long, created on November 11, 2020, and is hereby incorporated by reference. Background Technology

[0006] Soluble peptide-MHC I protein constructs have been previously described. These constructs can be used for a variety of applications, including those targeting rodents (e.g., Rodents are immunized to produce anti-peptide-in-groove antibodies. However, better soluble peptide-MHC II protein constructs are needed. Summary of the Invention

[0007] Compositions comprising MHC ligand peptides covalently attached to MHC class II molecules, nucleic acids encoding such compositions, and methods for inducing an immune response in subjects using such compositions are provided.

[0008] In one aspect, a composition comprising an MHC ligand peptide covalently attached to an MHC class II molecule, said MHC class II molecule comprising an MHC class II α chain or a portion thereof and an MHC class II β chain or a portion thereof. In some such compositions, the MHC ligand peptide is covalently attached to the MHC class II molecule via a peptide linker. In some such compositions, the MHC ligand peptide or peptide linker comprises a first cysteine ​​residue and the MHC class II molecule comprises a second cysteine ​​residue. In some such compositions, the first cysteine ​​residue and the second cysteine ​​residue form a disulfide bond such that the MHC ligand peptide binds to a peptide-binding groove formed by the MHC class II α chain or a portion thereof and the MHC class II β chain or a portion thereof.

[0009] In some such compositions, the MHC class II α chain or a portion thereof comprises an α1 domain, and the MHC class II β chain or a portion thereof comprises a β1 domain. Optionally, the MHC class II α chain or a portion thereof comprises an MHC class II α chain extracellular domain, and the MHC class II β chain or a portion thereof comprises an MHC class II β chain extracellular domain. Optionally, the MHC class II α chain or a portion thereof comprises an α1 domain, an α2 domain, a transmembrane domain, and a cytoplasmic domain. Optionally, the MHC class II β chain or a portion thereof comprises a β1 domain, a β2 domain, a transmembrane domain, and a cytoplasmic domain.

[0010] In some such compositions, the composition is membrane-anchored. In some such compositions, the composition is soluble. Optionally, the MHC class II α chain or a portion thereof contains α1 and α2 domains, but not transmembrane or cytoplasmic domains. Optionally, the MHC class II β chain or a portion thereof contains β1 and β2 domains, but not transmembrane or cytoplasmic domains. Optionally, the MHC class II α chain or a portion thereof and the MHC class II β chain or a portion thereof are connected by Jun-Fos zippers, electrostatic engineering, knob-in-holes, immunoglobulin scaffolds, immunoglobulin Fc regions, or linkers. Optionally, the MHC class II α chain or a portion thereof and the MHC class II β chain or a portion thereof are linked by a Jun-Fos zipper comprising a Jun leucine zipper dimerization motif and a Fos leucine zipper dimerization motif, and the MHC class II α chain or a portion thereof is linked to the Jun leucine zipper dimerization motif and the MHC class II β chain or a portion thereof is linked to the Fos leucine zipper dimerization motif, or the MHC class II α chain or a portion thereof is linked to the Fos leucine zipper dimerization motif and the MHC class II β chain or a portion thereof is linked to the Jun leucine zipper dimerization motif. Optionally, the C-terminus of the MHC class II α chain or a portion thereof is linked to the Jun leucine zipper dimerization motif, and the C-terminus of the MHC class II β chain or a portion thereof is linked to the Fos leucine zipper dimerization motif. Optionally, the C-terminus of the MHC class II α chain or a portion thereof is linked to a Fos leucine zipper dimerization motif, and the C-terminus of the MHC class II β chain or a portion thereof is linked to a Jun leucine zipper dimerization motif. Optionally, the MHC class II α chain or a portion thereof is linked to a Jun leucine zipper dimerization motif via an MHC-Jun linker, and the MHC class II β chain or a portion thereof is linked to a Fos leucine zipper dimerization motif via an MHC-Fos linker. Optionally, the MHC-Jun linker and the MHC-Fos linker each comprise the sequence listed in SEQ ID NO:1.

[0011] In some of these compositions, the MHC ligand peptide is about 10 to about 18 amino acids long, about 10 to about 15 amino acids long, or about 10 to about 12 amino acids long. In some of these compositions, the MHC ligand peptide is about 10 to about 18 amino acids long, about 10 to about 15 amino acids long, or about 10 to about 12 amino acids long. In some of these compositions, the MHC ligand peptide contains residues P-1 to P9 or residues P-3 to P9. In some of these compositions, the MHC ligand peptide is an antigenic MHC ligand peptide. In some of these compositions, the MHC ligand peptide is associated with T cell-mediated diseases.

[0012] In some of these compositions, the peptide linker connecting the MHC ligand peptide to the MHC class II molecule is a flexible linker. In some of these compositions, the peptide linker connecting the MHC ligand peptide to the MHC class II molecule comprises one or more flexible amino acids and one or more polar amino acids. In some of these compositions, the peptide linker connecting the MHC ligand peptide to the MHC class II molecule does not contain any charged amino acids. In some of these compositions, the peptide linker connecting the MHC ligand peptide to the MHC class II molecule includes a cleavage site. Optionally, the cleavage site is a tobacco etch virus (TEV) protease cleavage site.

[0013] In some of these compositions, the peptide linker connecting the MHC ligand peptide to the MHC class II molecule is non-immunogenic. In some of these compositions, the peptide linker connecting the MHC ligand peptide to the MHC class II molecule is attached to the N-terminus of the MHC class II β chain or a portion thereof. In some of these compositions, the peptide linker connecting the MHC ligand peptide to the MHC class II molecule is attached to the N-terminus of the MHC class II α chain or a portion thereof. In some of these compositions, the length of the peptide linker connecting the MHC ligand peptide to the MHC class II molecule is at least about 9 amino acids. In some of these compositions, the length of the peptide linker connecting the MHC ligand peptide to the MHC class II molecule is at least 9 amino acids. In some of these compositions, the length of the peptide linker connecting the MHC ligand peptide to the MHC class II molecule is between about 9 and about 50 amino acids. In some of these compositions, the length of the peptide linker connecting the MHC ligand peptide to the MHC class II molecule is between 9 and 50 amino acids. In some of these compositions, the peptide linker connecting the MHC ligand peptide to the MHC class II molecule contains 2-4 repeats of the sequence listed in SEQ ID NO:4.

[0014] In some such compositions, the peptide linker connecting the MHC ligand peptide to the MHC class II molecule comprises a first cysteine. Optionally, the first cysteine ​​is the only cysteine ​​in the peptide linker connecting the MHC ligand peptide to the MHC class II molecule. Optionally, the first cysteine ​​is located in the first four amino acids of the peptide linker connecting the MHC ligand peptide to the MHC class II molecule. In some such compositions, the peptide linker connecting the MHC ligand peptide to the MHC class II molecule comprises 2-4 repeats of the sequence listed in SEQ ID NO:4, wherein one amino acid in one of the repeats is mutated to a cysteine. Optionally, the peptide linker connecting the MHC ligand peptide to the MHC class II molecule comprises the sequence listed in SEQ ID NO:21.

[0015] In some such compositions, the MHC ligand peptide comprises a first cysteine. Optionally, the first cysteine ​​is opposite to the epitope formed by the composition.

[0016] In some such compositions, the second cysteine ​​residue is located in the MHC class II α chain or a portion thereof. Optionally, the MHC ligand peptide is linked to the peptide linker of the MHC class II molecule to the N-terminus of the MHC class II β chain or a portion thereof.

[0017] In some such compositions, a second cysteine ​​is absent in the wild-type MHC class II molecule corresponding to the MHC class II molecule in the composition. Optionally, the second cysteine ​​replaces a non-cysteine ​​amino acid in the corresponding wild-type MHC class II molecule. Optionally, the second cysteine ​​is located in the MHC class II α chain or a portion thereof. Optionally, when the MHC class II α chain or a portion thereof is best aligned with SEQ ID NO:49, the second cysteine ​​is located at position 101 in the sequence listed in SEQ ID NO:49. For example, the second cysteine ​​may be a... Figure 3 The alignment of the full-length sequences of HLA-DPA1, HLA-DQA1, and HLA-DRA1 marks the position corresponding to the DQA1 R101. For example, when the MHC class II α chain or a portion thereof is best aligned with SEQ ID NO:59, the second cysteine ​​residue in the corresponding wild-type MHC class II α chain can be located at the position corresponding to position 78 in the sequence listed in SEQ ID NO:59.

[0018] In some such compositions, the MHC class II molecule lacks the cysteine ​​present in the corresponding wild-type MHC class II molecule. Optionally, the cysteine ​​present in the corresponding wild-type MHC class II molecule has been replaced by any other amino acid. Optionally, the cysteine ​​present in the corresponding wild-type MHC class II molecule has been replaced by alanine, glutamine, tryptophan, or arginine. Optionally, the cysteine ​​present in the corresponding wild-type MHC class II molecule has been replaced by alanine or glutamine in the MHC class II molecule of the composition.

[0019] In some such compositions, the MHC class II α chain or a portion thereof lacks the cysteine ​​present in the corresponding wild-type MHC class II α chain. Optionally, the cysteine ​​present in the corresponding wild-type MHC class II α chain has been replaced by alanine or glutamine in the MHC class II α chain or a portion thereof in the composition. Optionally, when the MHC class II α chain or a portion thereof is best aligned with SEQ ID NO:49, the cysteine ​​in the corresponding wild-type MHC class II α chain is located at position 70 in the sequence listed in SEQ ID NO:49. For example, the cysteine ​​in the corresponding wild-type MHC class II α chain may be located at position 70 in the sequence listed in SEQ ID NO:49. Figure 3 The alignment of the full-length HLA-DPA1, HLA-DQA1, and HLA-DRA1 sequences marks the position corresponding to the DQA1 C70 position. For example, when the MHC class II α chain or a portion thereof is best aligned with SEQ ID NO:59, the cysteine ​​residue in the corresponding wild-type MHC class II α chain can be located at position 47 in the sequence listed in SEQ ID NO:59.

[0020] In some such compositions, the composition further comprises one or more immunostimulatory molecules. Optionally, the one or more immunostimulatory molecules are T cell epitopes that induce a T cell-mediated immune response to the composition. Optionally, the one or more immunostimulatory molecules comprise a pan-DR binding epitope (PADRE) and / or a peptide derived from lymphocytic choriomeningitis virus (LCMV). Optionally, the one or more immunostimulatory molecules are directly or indirectly covalently linked to an MHC class II molecule. Optionally, the one or more immunostimulatory molecules are directly or indirectly covalently linked to a portion of the MHC class II α chain or / or a portion of the MHC class II β chain or / or a portion of the MHC class II β chain.

[0021] In some of these compositions, the MHC class II molecule is a human MHC class II molecule. Optionally, the human MHC class II molecule is selected from the group consisting of HLA-DQ, HLA-DR, and HLA-DP. Optionally, the human MHC class II molecule is an HLA-DQ2 molecule. Optionally, the human MHC class II molecule is an HLA-DR2 molecule.

[0022] In some of these compositions, the MHC class II α chain or a portion thereof contains an MHC class II α chain extracellular domain, and the MHC class II β chain or a portion thereof contains an MHC class II β chain extracellular domain. The peptide linker connecting the MHC ligand peptide to the MHC class II molecule is a flexible linker of length between about 9 and about 50 amino acids. The linker contains a first cysteine ​​residue and is attached to the N-terminus of the MHC class II β chain or a portion thereof. A second cysteine ​​residue is located in the MHC class II α chain or a portion thereof and is absent in the wild-type MHC class II molecule corresponding to the MHC class II molecule in the composition. The MHC class II molecule lacks the cysteine ​​residue present in the corresponding wild-type MHC class II molecule. In some such compositions, the MHC class II α chain or a portion thereof contains an MHC class II α chain extracellular domain, and the MHC class II β chain or a portion thereof contains an MHC class II β chain extracellular domain. The peptide linker connecting the MHC ligand peptide to the MHC class II molecule is a flexible linker of length between 9 and 50 amino acids. The linker contains a first cysteine ​​residue and is attached to the N-terminus of the MHC class II β chain or a portion thereof. A second cysteine ​​residue is located in the MHC class II α chain or a portion thereof and is absent in the wild-type MHC class II molecule corresponding to the MHC class II molecule in the composition. The MHC class II molecule lacks the cysteine ​​residue present in the corresponding wild-type MHC class II molecule. Optionally, the composition is soluble, the MHC class II α chain or a portion thereof contains α1 and α2 domains but not transmembrane or cytoplasmic domains, the MHC class II β chain or a portion thereof contains β1 and β2 domains but not transmembrane or cytoplasmic domains, and the MHC class II α chain or a portion thereof and the MHC class II β chain or a portion thereof are linked by a Jun-Fos zipper containing a Jun leucine zipper dimerization motif and a Fos leucine zipper dimerization motif. Optionally, when the MHC class II α chain or a portion thereof is best aligned with SEQ ID NO:49, the second cysteine ​​is located at position 101 in the sequence listed in SEQ ID NO:49, and when the MHC class II α chain or a portion thereof is best aligned with SEQ ID NO:49, the cysteine ​​in the corresponding wild-type MHC class II molecule is located at position 70 in the sequence listed in SEQ ID NO:49. For example, the second cysteine ​​may be... Figure 3 The alignment of the full-length HLA-DPA1, HLA-DQA1, and HLA-DRA1 sequences in the datasets corresponds to the position marked DQA1 R101, and the cysteine ​​residues in the corresponding wild-type MHC class II α chain can be located at positions corresponding to... Figure 3The alignment of the full-length sequences of HLA-DPA1, HLA-DQA1, and HLA-DRA1 marks the position corresponding to the position of DQA1 C70. For example, when the MHC class II α chain or a portion thereof is best aligned with SEQ ID NO:59, the second cysteine ​​residue in the corresponding wild-type MHC class II α chain may be located at the position corresponding to position 78 in the sequence listed in SEQ ID NO:59, and when the MHC class II α chain or a portion thereof is best aligned with SEQ ID NO:59, the cysteine ​​residue in the corresponding wild-type MHC class II α chain may be located at the position corresponding to position 47 in the sequence listed in SEQ ID NO:59. Optionally, the MHC class II molecule is a human MHC class II molecule selected from the group consisting of HLA-DQ, HLA-DP, and HLA-DR. Optionally, the human MHC class II molecule is HLA-DQ.

[0023] Optionally, the extracellular domain of the MHC class II α chain comprises SEQ ID NO:64. Optionally, the extracellular domain of the MHC class II α chain is substantially composed of SEQ ID NO:64. Optionally, the extracellular domain of the MHC class II α chain is composed of SEQ ID NO:64. Optionally, the MHC class II α chain or a portion thereof (e.g., the extracellular domain of the MHC class II α chain) is attached (e.g., at the C-terminus) to a Fos leucine zipper dimerization motif. Optionally, the Fos leucine zipper dimerization motif comprises SEQ ID NO:23. Optionally, the Fos leucine zipper dimerization motif is substantially composed of SEQ ID NO:23. Optionally, the Fos leucine zipper dimerization motif is composed of SEQ ID NO:23. Optionally, the extracellular domain of the MHC class II β chain comprises SEQ ID NO:60. Optionally, the extracellular domain of the MHC class II β chain is substantially composed of SEQ ID NO:60. Optionally, the extracellular domain of the MHC class II β chain is composed of SEQ ID NO:60. Optionally, the MHC class II β chain or a portion thereof (e.g., the extracellular domain of the MHC class II β chain) is linked (e.g., at the C-terminus) to the Jun leucine zipper dimer motif. Optionally, the Jun leucine zipper dimer motif comprises SEQ ID NO:24. Optionally, the Jun leucine zipper dimer motif is substantially composed of SEQ ID NO:24. Optionally, the Jun leucine zipper dimer motif is composed of SEQ ID NO:24. Optionally, the extracellular domain of the MHC class II β chain is linked to the Jun leucine zipper dimer motif via a linker. Optionally, the linker comprises SEQ ID NO:1. Optionally, the linker is substantially composed of SEQ ID NO:1. Optionally, the linker is composed of SEQ ID NO:1. Optionally, the N-terminus of the MHC class II β chain or a portion thereof (e.g., the extracellular domain of the MHC class II β chain) is linked to an MHC ligand peptide (e.g., the C-terminus of the MHC ligand peptide) via a linker. Optionally, the linker comprises SEQ ID NO:21. Optionally, the linker consists essentially of SEQ ID NO:21. Optionally, the linker consists of SEQ ID NO:21. Optionally, the MHC ligand peptide is about 10 to about 18 amino acids in length, about 10 to about 15 amino acids in length, or about 10 to about 12 amino acids in length, and / or optionally, the MHC ligand peptide comprises residues P-1 to P9 or residues P-3 to P9. Optionally, the MHC ligand peptide is 10 to 18 amino acids in length, about 10 to 15 amino acids in length, or about 10 to 12 amino acids in length, and / or optionally, the MHC ligand peptide comprises residues P-1 to P9 or residues P-3 to P9.

[0024] On the other hand, nucleic acids encoding any of the above compositions are provided.

[0025] On the other hand, methods for inducing an immune response in subjects are provided. Some of these methods involve administering an effective amount of any of the above-described compositions or nucleic acids encoding the compositions to the subject.

[0026] In another aspect, methods for generating antigen-binding proteins are provided. Some such methods include: (a) immunizing a non-human animal with any of the above-described compositions or a nucleic acid encoding the composition; and (b) maintaining the non-human animal under conditions sufficient to induce an immune response to the composition. Optionally, the antigen-binding protein specifically binds to the antigenic composition, said antigenic composition comprising an MHC ligand peptide covalently attached to an MHC class II molecule. In some embodiments, the antigen-binding protein is an immunoglobulin molecule or a fragment thereof. In some embodiments, the antigen-binding protein is a T-cell receptor molecule or a fragment thereof. In another aspect, methods for generating antigen-binding proteins that specifically bind to antigenic compositions, said antigenic compositions comprising an MHC ligand peptide covalently attached to an MHC class II molecule are provided. Some such methods include: (a) immunizing a non-human animal with any of the above-described compositions or a nucleic acid encoding the composition; and (b) maintaining the non-human animal under conditions sufficient to induce an immune response to the composition. In some embodiments, the antigen-binding protein is an immunoglobulin molecule or a fragment thereof. In some embodiments, the antigen-binding protein is a T-cell receptor molecule or a fragment thereof. Attached Figure Description

[0027] Figure 1 (Not to scale) Some embodiments of various soluble peptide-MHC II constructs are shown. The sequence (SGGGGG) used in some constructs to link the Fos and Jun leucine zipper dimerization motifs to the α and β chains is listed in SEQ ID NO:1. The marker indicates a cysteine ​​residue engineered into the linker (linker Cys) and α chain (R101C) of construct B for disulfide stapling of the peptide. The marker also indicates a mutation of the cysteine ​​residue at position 70 in the α chain to glutamine (C70Q) or alanine (C70A). The asterisk indicates Davis-body modification (CH3 modification that allows differential binding of Fc to protein A).

[0028] Figure 2 The comparisons are shown for full-length DQ2α chain segments that do not contain mutations, contain C70Q mutations, or contain R101C and C70A mutations.

[0029] Figure 3The comparison shows the full-length α-chain segments from different HLA class II alleles.

[0030] Figure 4 The results from a Biacore assay show that, in some embodiments, soluble construct C binds to an anti-class II monoclonal antibody captured on the surface of an anti-mFc sensor.

[0031] Figure 5 The results from Biacore assays show that, in some embodiments, soluble construct C captured on the surface of the anti-hFc sensor binds to an anti-class II monoclonal antibody.

[0032] Figure 6A and 6B As shown, in some embodiments, a soluble construct in which the peptide is tethered to an HLA-DQB chain and the HLAα and β chains are dimerized in a Jun / Fos or Fc knob-pore arrangement is illustrated.

[0033] definition

[0034] The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, encompass amino acids of any length in polymeric form, including coding and non-coding amino acids, as well as amino acids modified or derived chemically or biochemically. These terms also include modified polymers, such as polypeptides having a modified peptide backbone. The term “domain” refers to any portion of a protein or polypeptide that has a specific function or structure.

[0035] Proteins are described as having an "N-terminus" (amino terminus) and a "C-terminus" (carboxy or carboxyl terminus). The term "N-terminus" refers to the starting point of a protein or polypeptide that terminates at an amino acid with a free amine group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (protein or polypeptide) that terminates at a free carboxyl group (-COOH).

[0036] The terms “nucleic acid” and “polynucleotide”, used interchangeably herein, encompass nucleotides of any length in polymeric form, including ribonucleotides, deoxyribonucleotides, or their analogues or modified forms. These nucleotides comprise single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derived nucleotide bases.

[0037] Nucleic acids are considered to have both a "5' end" and a "3' end" because mononucleotides react to form oligonucleotides in such a way that the 5' phosphate of one mononucleotide's pentose ring attaches to the 3' oxygen of its adjacent mononucleotide's pentose ring in one direction via a phosphodiester bond. If the 5' phosphate of the oligonucleotide is not attached to the 3' oxygen of the mononucleotide's pentose ring, then the end of the oligonucleotide is called a "5' end." If the 3' oxygen of the oligonucleotide is not attached to the 5' phosphate of another mononucleotide's pentose ring, then the end of the oligonucleotide is called a "3' end." Even if the nucleic acid sequence is inside a larger oligonucleotide, the nucleic acid sequence can be considered to have both a 5' end and a 3' end. In linear or circular DNA molecules, discrete elements are referred to as "downstream" or "upstream" of 3' elements or 5' elements.

[0038] The terms "expression vector," "expression construct," or "expression cassette" refer to a recombinant nucleic acid containing a desired coding sequence operatively linked to an appropriate nucleic acid sequence necessary for expression of the operatively linked coding sequence in a specific host cell or organism. The nucleic acid sequence necessary for expression in prokaryotes typically includes a promoter, an operon (optional), a ribosome binding site, and other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals, but some elements can be deleted and others added without sacrificing necessary expression.

[0039] A promoter is a regulatory region of DNA that typically includes a TATA box that guides RNA polymerase II to initiate RNA synthesis at the appropriate transcription start site of a specific polynucleotide sequence.

[0040] In some embodiments of the invention, the promoter may additionally include other regions that influence the transcription initiation rate. The promoter sequences disclosed herein in some embodiments regulate the transcription of operatively linked polynucleotides. The promoter may be active in one or more cell types disclosed herein in some embodiments (e.g., but not limited to, eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, single-cell embryos, differentiated cells, or combinations thereof). The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a time-restricted promoter (e.g., but not limited to, developmentally regulated promoters), or a spatially restricted promoter (e.g., but not limited to, cell-specific or tissue-specific promoters).

[0041] "Operationally connected" or "operably linked" includes juxtaposing two or more components (e.g., but not limited to, a promoter and another sequence element) such that both components function normally and that at least one component mediates the function imposed on at least one of the other components. As a non-limiting example, a promoter may be operably linked to a coding sequence if it controls the transcriptional level of a coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. An operational connection may include sequences that are adjacent to each other or act in a trans-regulatory manner (e.g., but not limited to, regulatory sequences that act at a distance to control the transcription of a coding sequence).

[0042] The term "isolated" in relation to proteins, nucleic acids, and cells includes proteins, nucleic acids, and cells that are relatively purified relative to other cellular or biological components that can normally be present in situ, up to and including substantially pure preparations containing proteins, nucleic acids, or cells.

[0043] In some embodiments of the invention, the term "isolated" may include proteins and nucleic acids that do not have naturally occurring counterparts, or proteins or nucleic acids that have been chemically synthesized and are therefore substantially uncontaminated by other proteins or nucleic acids. The term "isolated" may also include proteins, nucleic acids, or cells that have been isolated or purified from most other cellular or biological components (e.g., but not limited to other cellular proteins, nucleic acids, or cellular or extracellular components) that naturally accompany proteins, nucleic acids, or cells.

[0044] “Codon optimization” takes advantage of the degeneracy of codons, as demonstrated by the diversity of tribase pair codon combinations for a given amino acid, and typically involves modifying a nucleic acid sequence to enhance expression in a specific host cell by replacing at least one codon of the natural sequence with a codon that is more frequently or most frequently used in the host cell’s gene while maintaining the natural amino acid sequence. As a non-limiting example, nucleic acids encoding proteins can be modified to replace codons that have a higher frequency of use in a given prokaryotic or eukaryotic cell, including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, or any other host cell, compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, in “codon usage databases.” These tables can be modified in various ways. See Nakamura et al., (2000) Nucleic Acids Research, 28(1):292, which is incorporated herein by reference in its entirety for all purposes. Computer algorithms for codon optimization of specific sequences expressed in a particular host are also available (see, for example, Gene Forge).

[0045] The term "locus" refers to a specific location on a chromosome of an organism's genome, including a gene (or significant sequence), a DNA sequence, a polypeptide coding sequence, or a gene. As a non-limiting example, an "HLA locus" can refer to a specific location on an HLA gene, an HLA DNA sequence, an HLA coding sequence, or an HLA location on a chromosome of an organism's genome, where the location has been identified as the location where this sequence resides. An "HLA locus" may include regulatory elements of an HLA gene, including, for example, enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), or combinations thereof.

[0046] The term "gene" refers to a DNA sequence in a chromosome, which, if naturally occurring, may contain at least one coding region and at least one non-coding region. The DNA sequence of a chromosome encoding a product (e.g., but not limited to RNA products and / or polypeptide products) may contain a coding region interrupted by non-coding introns and a sequence located adjacent to the coding region at both the 5' and 3' ends such that the gene corresponds to a full-length mRNA sequence (containing 5' and 3' untranslated sequences). Additionally, other non-coding sequences, including regulatory sequences (e.g., but not limited to promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequences, and matrix attachment regions, may be present in the gene. These sequences may be located near the gene's coding region (e.g., but not limited to within 10 kb) or at distant sites, and these sequences may influence the level or rate of gene transcription and translation.

[0047] The term "allele" refers to a variant form of a gene. Some genes have multiple different forms, located at the same location on a chromosome or at a genetic locus. Diploid organisms have two alleles at each locus. Each pair of alleles represents the genotype at a specific locus. If there are two identical alleles at a particular locus, the genotype is described as homozygous; if the two alleles are different, the genotype is described as heterozygous.

[0048] The methods and compositions provided herein employ a variety of different components. Some components throughout the specification may have active variants and fragments. Such components include, for example, MHC class II molecules. The biological activity of each of these components is described elsewhere herein. The term “functional” refers to the innate ability of a protein or nucleic acid (or a fragment or variant thereof) to exhibit biological activity or function. Such biological activity or function may include, for example, the ability of an MHC class II molecule to bind to MHC ligand peptides and / or to bind to T cell receptors (TCRs) and achieve a T cell response. The biological function of a functional fragment or variant may be the same as or substantially altered (e.g., but not limited to, specificity or selectivity or efficacy) compared to the original molecule, but retains the essential biological function of the molecule.

[0049] The term "wildtype" includes entities that have structures (e.g., but not limited to, nucleotide or amino acid sequences) found in a normal state or condition (compared to mutations, diseases, alterations, etc.). Wildtype genes and polypeptides often exist in a variety of different forms (e.g., alleles).

[0050] The term "variant" refers to a nucleotide sequence that differs from the most common sequence in the population (e.g., but not limited to, differing by one nucleotide) or a protein sequence that differs from the most common sequence in the population (e.g., but not limited to, differing by one amino acid).

[0051] When referring to proteins, the term "fragment" means a protein that is shorter than a full-length protein or has fewer amino acids. When referring to nucleic acids, the term "fragment" means a nucleic acid that is shorter than a full-length nucleic acid or has fewer nucleotides. Non-limiting examples of protein fragments may include N-terminal fragments (i.e., a portion of the C-terminus of a protein removed), C-terminal fragments (i.e., a portion of the N-terminus of a protein removed), or internal fragments (i.e., a portion of the internal portion of a protein removed).

[0052] In the context of two polynucleotide or polypeptide sequences, "sequence identity" or "identity" refers to the same residues in two sequences when compared for maximum correspondence within a specified comparison window. When using a percentage of sequence identity for proteins, dissimilar residue positions are typically distinguished by conserved amino acid substitutions, where an amino acid residue is substituted by another amino acid residue with similar chemical properties (e.g., but not limited to, charge or hydrophobicity) and therefore does not alter the functional properties of the molecule. When the conserved substitutions of sequences differ, the percentage sequence identity can be adjusted upwards to correct for the conservatism of the substitution. Thus, sequences that differ despite similar conserved substitutions are considered to have "sequence similarity" or "identity." The methods used to make such adjustments are well known. Typically, this involves counting conserved substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, as a non-limiting example, given a score of 1 for identical amino acids and a score of zero for non-conservative substitutions, the score for conserved substitutions is given as being between zero and 1. For example, scores for conservative substitution are calculated through the implementation of the project PC / GENE (Intelligenetics, Mountain View, California).

[0053] The "Sequence Identity Percentage" comprises a value (the maximum number of perfectly matched residues) determined by comparing two optimally aligned sequences within a comparison window. This comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (excluding additions or deletions) to achieve optimal alignment of the two sequences. The number of matching positions is calculated by determining the percentage of positions where the same nucleic acid base or amino acid residue appears in both sequences. This number of matching positions is divided by the total number of positions in the comparison window, and the result is multiplied by 100 to obtain the sequence identity percentage. Unless otherwise specified (e.g., the shorter sequence contains linked heterologous sequences), the comparison window is the full length of the shorter of the two compared sequences.

[0054] Unless otherwise stated, sequence identity / similarity values ​​include values ​​obtained using GAP version 10 with the following parameters: the percentage of identity and similarity of nucleotide sequences using a GAP weight of 50 and a length weight of 3 and an nwsgapdna.cmp scoring matrix; the percentage of identity and similarity of amino acid sequences using a GAP weight of 8 and a length weight of 2 and a BLOSUM62 scoring matrix; or any equivalent procedure thereof. An “equivalent procedure” includes any sequence comparison procedure that generates alignments with identical nucleotide or amino acid residue matches and the same percentage of sequence identity for any two sequences in question when compared to corresponding alignments generated by GAP version 10.

[0055] The term "in vitro" includes an artificial environment and processes or reactions that occur within that artificial environment (e.g., but not limited to, test tubes or isolated cells or cell lines). The term "in vivo" includes a natural environment (e.g., but not limited to, an organism or body or cells or tissues within an organism or body) and processes or reactions that occur within that natural environment. The term "ex vivo" includes cells that have been removed from an individual and processes or reactions that occur within such cells.

[0056] The terms “major histocompatibility complex” and “MHC” encompass the terms “human leukocyte antigen” or “HLA” (the latter two are generally reserved for human MHC molecules), naturally occurring MHC molecules, individual chains of MHC molecules (e.g., but not limited to, MHC class I α (heavy) chain, β2 microglobulin, MHC class II α chain and MHC class II β chain), individual subunits of such chains of MHC molecules (e.g., but not limited to, α1, α2 and / or α3 subunits of MHC class I α chain, α1-α2 subunits of MHC class II α chain, β1-β2 subunits of MHC class II β chain) and their portions (e.g., but not limited to, peptide-binding portions, such as peptide-binding grooves), mutants and various derivatives (including fusion proteins), wherein such portions, mutants and derivatives retain the ability to exhibit antigenic peptides to be recognized by T cell receptors (TCRs) (e.g., but not limited to, antigen-specific TCRs). MHC class I molecules contain a peptide-binding groove formed by the α1 and α2 domains of the heavy α chain, which can hold peptides of approximately 8–10 amino acids. Although both classes of MHC bind a core of approximately 9 amino acids (e.g., but not limited to 5–17 amino acids) within the peptide, the open-terminal nature of the MHC class II peptide-binding groove (the α1 domain of a class II MHC α peptide associated with the β1 domain of a class II MHC β peptide) allows for a wider range of peptide lengths. The length of MHC class II-binding peptides typically varies between 13 and 17 amino acids, but shorter or longer lengths are not uncommon. Therefore, peptides can translocate within the MHC class II peptide-binding groove, thereby altering the decimator sequence directly located within the groove at any given time. This article uses routine identification of specific MHC variants.

[0057] The term "antigen" refers to any agent (e.g., but not limited to, proteins, peptides, polysaccharides, glycoproteins, glycolipids, nucleotides, parts thereof, or combinations thereof) that, when introduced into an immune-active host, is recognized by the host's immune system and elicits an immune response. The TCR recognizes peptides present in the background of the MHC as part of an immune synapse. Peptide-MHC (pMHC) complexes are recognized by the TCR and possess both peptide (antigenic determinant) and TCR idiotypes, thus providing specificity for the interaction. Therefore, the term "antigen" encompasses peptides present in the background of the MHC (e.g., but not limited to, peptide-MHC complexes or pMHC complexes). Peptides displayed on the MHC may also be referred to as "epitopes" or "antigenic determinants." The terms "peptide," "antigenic determinant," "epitope," etc., encompass not only those naturally presented by antigen-presenting cells (APCs) but also any desired peptide, provided it is recognized by immune cells (e.g., but not limited to, when appropriately presented to cells of the immune system).

[0058] The terms “peptide-MHC class II complex,” “pMHC class II complex,” and “in-groove peptide” include (i) MHC class II molecules (e.g., but not limited to, human MHC class II molecules) or portions thereof (e.g., but not limited to, their peptide binding grooves or their extracellular portions), and (ii) antigenic peptides, wherein the MHC class II molecules and antigenic peptides are compounded in such a way that the pMHC class II complex can specifically bind to T cell receptors. The pMHC class II complex encompasses cell surface-expressed pMHC class II complexes and soluble pMHC class II complexes. Upon administration of an antigenic pMHC class II complex (e.g., but not limited to, a complex comprising an MHC class II molecule compounded with a peptide, such as a peptide exogenous to the animal to which the pMHC class II complex is administered), the animal is able to generate an antibody response to the antigenic pMHC class II complex and / or a T cell response to the antigenic pMHC class II complex (i.e., the generation of T cell receptors specific to the pMHC class II complex). Such specific antigen-binding proteins can then be isolated and used as therapeutic agents to specifically modulate interactions with specific T-cell receptors on antigenic pMHC class II complexes. Although in some cases, soluble pMHC class II complexes containing peptides complexed with MHC class II molecules (e.g., but not limited to, those exogenous to the host animal to which the pMHC class II complex is administered) may not elicit a T-cell immune response due to the soluble nature of the administered pMHC class II complex, such soluble pMHC class II complexes can still be considered antigenic because they can elicit a B-cell-mediated immune response that produces antigen-binding proteins that specifically bind to the soluble pMHC class II complex.

[0059] As used herein, the term "effective amount" includes the amount (e.g., but not limited to, sufficient to elicit or modulate an immune response) that effectively achieves the desired outcome within the necessary dose and time period. The effective amount of a peptide-MHC class II complex can vary depending on factors such as the subject's disease state, age, and weight, as well as the peptide-MHC class II complex's ability to elicit the desired response in the subject. Dosing regimens can be adjusted to provide the optimal response. An effective amount is also the amount in which any beneficial therapeutic effect outweighs any toxic or harmful effects of the peptide-MHC class II complex (e.g., but not limited to, side effects).

[0060] "Optional" or "optionally" means that the event or situation described below may or may not occur, and this description includes instances where the event or situation occurs and instances where the event or situation does not occur.

[0061] The specification of a numerical range includes all integers within the range or defining the range, as well as all subranges defined by the integers within the range.

[0062] Unless the context otherwise requires, the term “about” covers a value ±5.

[0063] The term “and / or” means and covers any and all possible combinations of one or more of the listed items, as well as the absence of combinations when interpreted in an alternative scheme (“or”).

[0064] The term "or" refers to any one of the members in a particular list.

[0065] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein include plural references. For example, the term “protein” or “at least one protein” can include multiple proteins, including mixtures thereof.

[0066] Statistically significant means p≤0.05. Detailed Implementation

[0067] I. Overview

[0068] This document provides compositions comprising MHC ligand peptides covalently attached to MHC class II molecules. In some embodiments of the invention, the MHC class II molecule may comprise an MHC class II α chain or a portion, fragment, or variant thereof, and an MHC class II β chain or a portion, fragment, or variant thereof. In some embodiments of the composition, the MHC ligand peptide is covalently attached to the MHC class II molecule via a peptide linker. The MHC ligand peptide or peptide linker may comprise a first cysteine ​​residue, and the MHC class II α chain or a portion, fragment, or variant thereof, or the MHC class II β chain or a portion, fragment, or variant thereof, may comprise a second cysteine ​​residue. In some embodiments, the first and second cysteine ​​residues may then form a disulfide bond, such that the MHC ligand peptide binds to a peptide-binding groove formed by the MHC class II α chain or a portion, fragment, or variant thereof, and the MHC class II β chain or a portion, fragment, or variant thereof. Nucleic acids encoding such compositions and methods for inducing an immune response in subjects using such compositions are also provided.

[0069] Soluble peptide-MHC I protein constructs have been previously described. These constructs can be used for a variety of applications, including, but not limited to, rodents (e.g., but not limited to, In rodents, immunization is used to generate antibodies against in-groove peptides or to produce T-cell receptors specific to peptide-MHC I proteins. We have now designed a peptide-MHC II protein construct in which the α and β chains of the MHC II molecule are anchored together and anchored to peptides in their grooves. These can be used for a variety of applications, such as, but not limited to, generating soluble MHC II constructs to act as immunogens, and generating membrane-anchored MHC II proteins for other applications, including recruiting T cells expressing MHC II class-peptide-specific TCRs.

[0070] II. Compositions containing peptide-MHC class II complexes

[0071] In some embodiments of the invention, various peptide-MHC class II complexes (pMHC complexes) are provided. Antigenic peptide-MHC class II complexes can be used, for example, to generate pMHC-specific antigen-binding proteins. Some such complexes comprise an MHC ligand peptide covalently attached to an MHC class II molecule comprising an MHC class II α chain or a portion, fragment, or variant thereof, and an MHC class II β chain or a portion, fragment, or variant thereof. In some embodiments of the complex, the MHC ligand peptide is covalently attached to the MHC class II molecule via a peptide linker. The MHC ligand peptide or peptide linker may contain a first cysteine ​​residue, and the MHC class II molecule (e.g., but not limited to, an MHC class II α chain or a portion, fragment, or variant thereof, or an MHC class II β chain or a portion, fragment, or variant thereof) may contain a second cysteine ​​residue, wherein the first and second cysteine ​​residues form a disulfide bond such that the MHC ligand peptide binds in a peptide-binding groove formed by the MHC class II α chain or a portion, fragment, or variant thereof, and the MHC class II β chain or a portion, fragment, or variant thereof.

[0072] In some embodiments, an MHC class II molecule that can be used as part of an antigenic peptide-MHC class II complex may include at least a portion or fragment or variant of an MHC class II α chain and at least a portion or fragment or variant of an MHC class II β chain (e.g., but not limited to, at least a portion or fragment or variant of an extracellular domain of an MHC class II α chain and at least a portion or fragment or variant of an extracellular domain of an MHC class II β chain), such that the portion or fragment or variant of the MHC class II α chain and the portion or fragment or variant of the MHC class II β chain form a peptide-binding groove that can bind to an MHC ligand peptide. As a non-limiting example, MHC class II molecules that can be used as part of an antigenic peptide-MHC class II complex may include naturally occurring full-length MHC and individual chains of MHC (e.g., but not limited to, the MHC class II α chain and the MHC class II β chain), individual subunits of such chains of MHC (e.g., but not limited to, the α1-α2 subunits of the MHC class II α chain and the β1-β2 subunits of the MHC class II β chain, or the α1 subunit of the MHC class II α chain and the β1 subunit of the MHC class II β chain), as well as fragments, mutants, and derivatives thereof (including fusion proteins), wherein such fragments, mutants, and derivatives retain the ability to display antigenic determinants for recognition by antigen-specific T cell receptors (TCRs). MHC class II molecules and MHC class II molecules that can be used as part of an antigenic peptide-MHC class II complex are described in more detail elsewhere herein.

[0073] In some embodiments of the complex, at least one chain of an MHC class II molecule (e.g., but not limited to, an MHC class II α chain or a portion, fragment, or variant thereof, or an MHC class II β chain or a portion, fragment, or variant thereof) and an MHC ligand peptide are associated to form a fusion protein. As a non-limiting example, an MHC class II molecule (e.g., but not limited to, an MHC class II β chain or a portion, fragment, or variant thereof, or an MHC class II α chain or a portion, fragment, or variant thereof) and an MHC ligand peptide may be linked by a linker. The linking of the MHC class II molecule to the MHC ligand peptide, and suitable linkers for doing so, are described in more detail below.

[0074] In some embodiments of the complex, the MHC ligand peptide or the linker connecting the MHC ligand peptide to at least one chain (or a portion, fragment, or variant thereof) of an MHC class II molecule is attached via a disulfide bridge. A disulfide bridge is a disulfide bond extending between a pair of oxidized cysteine ​​residues. The connection of the MHC ligand peptide or the linker connecting the MHC ligand peptide to at least one chain (or a portion, fragment, or variant thereof) of an MHC class II molecule via a disulfide bridge is described in more detail below.

[0075] The peptide-MHC class II complexes disclosed herein in some embodiments may be membrane-bound or soluble. MHC class II molecules are naturally occurring membrane-anchored heterodimers. Hydrophobic transmembrane regions of the α and β chains facilitate the assembly of the heterodimers. Some of the peptide-MHC class II complexes described herein are membrane-bound. As a non-limiting example, such membrane-bound peptide-MHC class II complexes may comprise MHC class II molecules containing transmembrane domains or containing both transmembrane and cytoplasmic domains. As a non-limiting example, the MHC class II molecules in the complex may include an α chain containing a transmembrane domain or containing both transmembrane and cytoplasmic domains, and / or the MHC class II molecules may include a β chain containing a transmembrane domain or containing both transmembrane and cytoplasmic domains.

[0076] In some embodiments, the peptide-MHC class II complex may be soluble (i.e., non-membrane-bound). As a non-limiting example, such a soluble peptide-MHC class II complex may include MHC class II molecules that do not contain a transmembrane domain or do not contain both transmembrane and cytoplasmic domains. As a non-limiting example, the MHC class II molecules in the complex may include an α-chain that does not contain a transmembrane domain or does not contain both transmembrane and cytoplasmic domains, and / or the MHC class II molecules may include a β-chain that does not contain a transmembrane domain or does not contain both transmembrane and cytoplasmic domains.

[0077] In some embodiments, the soluble peptide-MHC class II complex may further comprise other components to stabilize chain pairing between the MHC class II α chain or a portion or fragment or variant thereof and the MHC class II β chain or a portion or fragment or variant thereof. In some embodiments, non-limiting examples of mechanisms for stabilizing chain pairing include Jun-Fos zipper linkage, immunoglobulin scaffold linkage, immunoglobulin Fc region linkage (e.g., but not limited to, immunoglobulin Fc hinge region), immunoglobulin Fc knob-pore mutation, electrostatic engineering such as immunoglobulin Fc charge mutation (including but not limited to charge reversal mutation), direct linkers (e.g., but not limited to covalent bonds, such as peptide linkers), or any combination thereof. Detailed descriptions and non-limiting examples of each of these mechanisms are provided elsewhere herein. However, any other suitable chain pairing method may be used.

[0078] A. MHC class II molecules

[0079] In some embodiments of the present invention, any suitable MHC class II molecule can be used in the peptide-MHC class II complexes described herein. MHC molecules are generally divided into two classes: class I and class II MHC molecules. MHC class II molecules or MHC class II proteins are heterodimeric membrane proteins comprising a non-covalently associated α chain and a β chain. The α chain has two extracellular domains (α1 and α2) and two intracellular domains (TM domain and CYT domain). The β chain has two extracellular domains (β1 and β2) and two intracellular domains (TM domain and CYT domain).

[0080] The domains of class II MHC molecules organize to form antigenic determinant binding sites (e.g., peptide-binding moieties or peptide-binding grooves) of the MHC molecule. A peptide-binding groove is a cavity-forming portion of the MHC protein in which a peptide (e.g., an antigenic determinant) can bind. The conformation of the peptide-binding groove can change upon peptide binding, allowing the appropriate alignment of amino acid residues important for the binding of the TCR to the peptide-MHC (pMHC) complex.

[0081] In some embodiments of peptide-MHC class II complexes, MHC class II molecules include portions, fragments, or variants of class II MHC chains sufficient to form peptide-binding grooves. The peptide-binding groove of a class II protein may contain portions, fragments, or variants of α1 and β1 domains capable of forming two β-sheets and two α-helices. A first portion of the α1 domain forms a first β-sheet, and a second portion of the α1 domain forms a first α-helix. A first portion of the β1 domain forms a second β-sheet, and a second portion of the β1 domain forms a second α-helix. X-ray crystal structures of class II proteins having peptides bound to the binding groove of the protein show that one or both ends of the binding peptide may protrude beyond the MHC protein. See, for example, Brown et al. (1993), Nature 364(6432):33-39, which is incorporated herein by reference in its entirety for all purposes. Thus, the ends of the α1 and β1α-helices of class II form open cavities such that the ends of the peptide bound to the binding groove are not embedded in the cavities.

[0082] Many human and mammalian MHCs are known. As non-limiting examples of some implementations, human MHC IIα or β peptides may be derived from α or β peptides encoding functional human HLA molecules from any of the HLA-DP, HLA-DQ, HLA-DR, HLA-DM, or HLA-DO loci or combinations thereof. A list of common HLA antigens and alleles, along with a brief description of HLA nomenclature, is described in Shankarkumar et al., “The Human Leukocyte Antigen (HLA) System,” *International Journal of Human Genetics* 4(2):91-103, (2004), which is incorporated herein by reference in its entirety for all purposes. Further information on HLA nomenclature and various HLA alleles can be found in Holdsworth et al. (2009) Tissue Antigens 73(2):95-170 and Marsh (2019) International Journal of Immunogenetics 46(5):346-418, each of which is incorporated herein by reference in its entirety for all purposes.

[0083] In one exemplary embodiment, MHC is a human MHC class II molecule, such as a human HLA molecule expressed on the cell surface selected from the group consisting of HLA-DP, HLA-DR, HLA-DQ, and any combination thereof. As a non-limiting example, the peptide-MHC class II complex may comprise one or more MHC class II α chains or their domains, portions, fragments, or variants (e.g., but not limited to, one or more human MHC class II α chains or their domains, portions, fragments, or variants). As a non-limiting exemplary embodiment, the class II α chain may be HLA-DPA, HLA-DQA, or HLA-DRA. Similarly, in some embodiments, the peptide-MHC class II complex may comprise one or more MHC class II β chains or their domains, portions, fragments, or variants (e.g., but not limited to, one or more human MHC class II β chains or their domains, portions, fragments, or variants). As a non-limiting exemplary embodiment, the class II β chain may be HLA-DPB, HLA-DQB, or HLA-DRB.

[0084] In some implementations, particular interest is found in polymorphic human HLA alleles known to be associated with a variety of human diseases, such as, but not limited to, human autoimmune diseases. Specific polymorphisms in HLA loci have been identified as being associated with the development of rheumatoid arthritis, type 1 diabetes, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, Graves' disease, systemic lupus erythematosus, celiac disease, Crohn's disease, ulcerative colitis, and other autoimmune conditions. See, for example, de Bakker (2006) *Nature Genetics* 38(10):1166-1172; Wong and Wen (2004) *Diabetologia* 47(9):1476-1487; Taneja and David (1998) *Journal of Clinical Research* 101(5):921-926; and the International MHC and Autoimmunity Genetics Network (2009) *Proceedings of the National Academy of Sciences of the United States of America* 106(44):18680-18685, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, human MHC II peptides may be derived from human HLA molecules known to be associated with specific diseases (e.g., but not limited to, autoimmune diseases).

[0085] In one exemplary embodiment, human MHC class II molecules (e.g., but not limited to, human MHC IIα and β polypeptides or portions, fragments, or variants thereof) are derived from human HLA-DR (e.g., but not limited to HLA-DR2). Typically, the HLA-DRα chain is monomorphic (e.g., the α chain of an HLA-DR protein is encoded by an HLA-DRA gene, such as the HLA-DRα*01 gene). On the other hand, the HLA-DRβ chain is polymorphic. Thus, HLA-DR2 comprises an α chain encoded by an HLA-DRA gene and a β chain encoded by the HLADR1β*1501 gene. This document covers any suitable HLA-DR sequence, such as polymorphic variants exhibited in human populations, sequences with one or more conserved or non-conserved amino acid modifications, etc.

[0086] In another exemplary embodiment, human MHC class II molecules (e.g., but not limited to, human MHC II α and β peptides or portions or fragments thereof or variants) are derived from human HLA-DQ (e.g., but not limited to, HLA-DQ2). HLA-DQ2 is a serogroup determined by antibody recognition of the β2 subset of the DQ β chain. The β chain of DQ is encoded by the HLA-DQB1 locus, and DQ2 is encoded by the HLA-DQB1*02 allele. This group contains two common alleles, DQB1*0201 and DQB1*0202. The DQ2 β chain combines with the α chain encoded by the genetically linked HLA-DQA1 allele to form a cis-haplotype isotype. These isotypes, nicknamed DQ2.2 and DQ2.5, are also encoded by the DQA1*0201 and DQA1*0501 genes, respectively. DQ2.5 is one of the most susceptibility factors for autoimmune diseases. DQ2.5 is typically encoded by a haplotype associated with a wide range of diseases, such as autoimmune diseases. This haplotype, HLA A1-B8-DR3-DQ2, is associated with diseases suspected to be involved in by HLA-DQ2. For example, DQ2 is directly involved in celiac disease.

[0087] In another embodiment, human MHC class II molecules (e.g., but not limited to, human MHC IIα and β polypeptides, or portions or fragments thereof, or variants thereof) may be encoded by nucleotide sequences of HLA alleles known to be associated with common human diseases, or portions or fragments thereof. Such HLA alleles include, but are not limited to, HLA-DRB1*0401, HLA-DRB1*0301, HLA-DQA1*0501, HLA-DQB1*0201, HLA-DRB1*1501, HLA-DRB1*1502, HLA-DQB1*0602, HLA-DQA1*0102, HLA-DQA1*0201, HLA-DQB1*0202, HLA-DQA1*0501, and combinations thereof. A summary of HLA allele / disease associations is provided in de Bakker (2006) Nature Genetics 38(10):1166-1172, which is incorporated herein by reference in its entirety for all purposes. Other non-restricted examples of HLA alleles associated with common diseases include B*0801 / DRB1*0301 / DQA1*0501 / DQB1*0201 (Graves' disease or myasthenia gravis), DRB1*1501 / DQB1*0602 (multiple sclerosis), DQA1*0102 (multiple sclerosis), C*0602 (psoriasis), DQA1*0201 / DQB1*0202 (DQ2.2) (celiac disease), DQA1*0501 / DQB1*0201 (DQ2.5) (celiac disease), DRB1*1501 (systemic lupus erythematosus (SLE)), DRB1*0301 (type 1 diabetes or SLE), and B*5701 (abacavir hypersensitivity).

[0088] In some embodiments, the MHC class II molecule that can be used as part of an antigenic peptide-MHC class II complex includes an MHC class II α chain or a portion or fragment or variant thereof and an MHC class II β chain or a portion or fragment or variant thereof (e.g., but not limited to, the extracellular domain of the MHC class II α chain or a portion or fragment or variant thereof and the extracellular domain of the MHC class II β chain or a portion or fragment or variant thereof), such that the MHC class II α chain and β chain (or a portion or fragment or variant thereof) form a peptide-binding groove that can bind to an MHC ligand peptide. As a non-limiting example, MHC class II molecules that can be used as part of an antigenic peptide-MHC class II complex include naturally occurring full-length MHC and individual chains of MHC (e.g., but not limited to, MHC class II α chain and MHC class II β chain), individual subunits of such chains of MHC (e.g., but not limited to, α1-α2 subunits of the MHC class II α chain and β1-β2 subunits of the MHC class II β chain, or α1 subunit of the MHC class II α chain and β1 subunit of the MHC class II β chain), and their portions, fragments, mutants, and various derivatives (including fusion proteins), wherein such portions, fragments, mutants, and derivatives retain the ability to exhibit antigenic determinants for recognition by antigen-specific TCRs. In one exemplary embodiment, the MHC class II α chain or its portion, fragment, or variant, and the MHC class II β chain or its portion, fragment, or variant, included in the peptide-MHC class II complex contain the regions or minimum regions required to form a peptide-binding groove for an MHC ligand peptide. In one exemplary embodiment, the MHC class II α chain or a portion, fragment, or variant thereof and the MHC class II β chain or a portion, fragment, or variant thereof included in the peptide-MHC class II complex are substantially composed of the region required to form a peptide-binding groove for an MHC ligand peptide or the minimum required region.

[0089] The MHC class II molecules in the peptide-MHC complexes disclosed herein in some embodiments may be membrane-bound or soluble. MHC class II molecules are naturally occurring membrane-anchored heterodimers. The hydrophobic transmembrane regions of the α and β chains facilitate the assembly of the heterodimers. Some MHC class II molecules in the peptide-MHC class II complexes disclosed herein in some embodiments are membrane-bound. As a non-limiting example, such membrane-bound peptide-MHC class II complexes may comprise MHC class II molecules containing a transmembrane domain or a portion, fragment, or variant thereof, or containing both transmembrane and cytoplasmic domains or a portion, fragment, or variant thereof. As a non-limiting example, the MHC class II molecules in the complex may include an α chain containing a transmembrane domain or a portion, fragment, or variant thereof, or containing both transmembrane and cytoplasmic domains or a portion, fragment, or variant thereof, and / or the MHC class II molecules may include a β chain containing a transmembrane domain or a portion, fragment, or variant thereof, or containing both transmembrane and cytoplasmic domains or a portion, fragment, or variant thereof.

[0090] In some embodiments, the MHC class II molecules in the peptide-MHC class II complexes disclosed herein may be soluble (i.e., not membrane-bound). In one exemplary embodiment, such soluble peptide-MHC class II complexes may comprise MHC class II molecules that do not contain a transmembrane domain or do not contain both transmembrane and cytoplasmic domains. In another exemplary embodiment, the MHC class II molecules in the complex may comprise an α chain or a portion or fragment thereof or a variant thereof that does not contain a transmembrane domain or does not contain both transmembrane and cytoplasmic domains, and / or the MHC class II molecules may comprise a β chain or a portion or fragment thereof or a variant thereof that does not contain a transmembrane domain or does not contain both transmembrane and cytoplasmic domains.

[0091] In one embodiment, the α chain, or a portion, fragment, or variant thereof, comprises a full-length α chain fragment that does not contain a transmembrane domain or a C-terminal region of a transmembrane domain at its C-terminus. In another embodiment, the α chain, or a portion, fragment, or variant thereof, comprises a full-length α chain fragment that does not include a signal peptide at its N-terminus and does not include a transmembrane domain or a C-terminal region of a transmembrane domain at its C-terminus. In a non-limiting example, the α chain, or a portion, fragment, or variant thereof, may be operatively linked to different signal peptides. In one exemplary embodiment, the α chain or a portion or fragment or variant thereof comprises amino acid residues 24-216 of SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:57, or a fragment of the full-length α chain corresponding to amino acid residues 24-216 of SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:57 (e.g., when the full-length α chain of the derived α chain or a portion or fragment or variant thereof is best aligned with SEQ ID NO:49, SEQ ID NO:53 or SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:57). In another embodiment, the α chain, or a portion or fragment thereof, or a variant thereof, comprises amino acid residues 29-222 of SEQ ID NO:51, or a segment of the full-length α chain corresponding to amino acid residues 29-222 of SEQ ID NO:51 (e.g., when the full-length α chain of the derived α chain, or a portion or fragment thereof, or a variant thereof, is best aligned with SEQ ID NO:51). In another embodiment, the α chain, or a portion or fragment thereof, or a variant thereof, comprises amino acid residues 26-216 of SEQ ID NO:52 or 58, or a segment of the full-length α chain corresponding to amino acid residues 26-216 of SEQ ID NO:52 or 58 (e.g., when the full-length α chain of the derived α chain, or a portion or fragment thereof, or a variant thereof, is best aligned with SEQ ID NO:52 or 58). In another embodiment, the α chain, or a portion or fragment thereof, or a variant thereof, comprises a sequence listed in any one of SEQ ID NO:59 and 61-68.

[0092] In one embodiment, the β chain, or a portion, fragment, or variant thereof, comprises a fragment of a full-length β chain that does not include a transmembrane domain or a C-terminal region of a transmembrane domain at its C-terminus. In another embodiment, the β chain, or a portion, fragment, or variant thereof, comprises a fragment of a full-length β chain that does not include a signal peptide at its N-terminus and does not include a transmembrane domain or a C-terminal region of a transmembrane domain at its C-terminus. In a non-limiting example, the β chain, or a portion, fragment, or variant thereof, may be operably linked to different signal peptides. In an exemplary embodiment, the β chain, or a portion, fragment, or variant thereof, comprises amino acid residues 33-230 of SEQ ID NO:50, or a fragment of the full-length β chain corresponding to amino acid residues 33-230 of SEQ ID NO:50 (e.g., when the full-length β chain of the derived β chain, or a portion, fragment, or variant thereof, is best aligned with SEQ ID NO:50). In another exemplary embodiment, the β chain, or a portion, fragment, or variant thereof, comprises the sequence listed in SEQ ID NO:60.

[0093] In some embodiments, the MHC II molecule in the soluble peptide-MHC class II complex may further include other components to stabilize chain pairing between the MHC class II α chain or a portion or fragment or variant thereof and the MHC class II β chain or a portion or fragment or variant thereof. In some embodiments, non-limiting examples of mechanisms for stabilizing chain pairing include Jun-Fos zipper connections, immunoglobulin scaffold connections, immunoglobulin Fc regions (e.g., but not limited to immunoglobulin Fc hinge regions), immunoglobulin Fc knob-pores, electrostatic engineering such as immunoglobulin Fc charge mutations (including but not limited to charge reversal mutations), direct linkers (e.g., but not limited to covalent bonds, such as peptide linkers), or any combination thereof. However, any other suitable chain pairing method may be used.

[0094] In one embodiment, an MHC class II α chain, or a portion or fragment or variant thereof, and an MHC class II β chain, or a portion or fragment or variant thereof, are linked by a Jun-Fos zipper. Synthetic peptides of known Fos and Jun leucine zipper dimerization motifs assemble into stable, soluble heterodimers. See, for example, Kalandadze et al. (1996), *J. Biol. Chem.* 271:20156-20162, and Gauthier et al. (1998), *Proceedings of the National Academy of Sciences* 95:11828-11833, each of which is incorporated herein by reference in its entirety for all purposes. The leucine zipper is characterized by a periodic interval of five leucine residues every seven residues (a heptapeptide repeat). Each heptapeptide repeat contributes two turns of the α-helix. The leucine residues have a specific function in the leucine zipper dimerization and form an interface between the two α-helices of the coiled helix. The Jun / Fos heterodimer is soluble due to the charged residues on the outer surface of the coiled helix. In one exemplary embodiment, the MHC class II α chain or a portion, fragment, or variant thereof and the MHC class II β chain or a portion, fragment, or variant thereof (e.g., but not limited to, the C-terminus of the MHC class II α chain or a portion, fragment, or variant thereof and the C-terminus of the MHC class II β chain or a portion, fragment, or variant thereof) may be linked to leucine zipper dimerization motifs from transcription factors Fos and Jun, assembling into a soluble, tightly packed coiled helical structure. In another exemplary embodiment, the hydrophobic transmembrane regions of the MHC class II α chain and the MHC class II β chain are replaced by leucine zipper dimerization motifs from transcription factors Fos and Jun. In another exemplary embodiment, the extracellular domains of the MHC class II α chain or a portion or fragment or variant thereof (e.g., but not limited to, containing the MHC class II α1 domain or a portion or fragment or variant thereof, or the MHC class II α1 and α2 domains or a portion or fragment or variant thereof) may be linked (e.g., but not limited to, in-frame fusion or via a linker) to the Fos leucine zipper dimer motif, and the extracellular domains of the MHC class II β chain or a portion or fragment or variant thereof (e.g., but not limited to, containing the MHC class II β1 domain or a portion or fragment or variant thereof, or the MHC class II β1 and β2 domains or a portion or fragment or variant thereof) may be linked (e.g., but not limited to, in-frame fusion or via a linker) to the Jun leucine zipper dimer motif.In another exemplary embodiment, the extracellular domains of the MHC class II α chain or a portion or fragment or variant thereof (e.g., but not limited to, containing the MHC class II α1 domain or a portion or fragment or variant thereof, or the MHC class II α1 and α2 domains or a portion or fragment or variant thereof) can be linked (e.g., but not limited to, in-frame fusion or via a linker) to the Jun leucine zipper dimer motif, and the extracellular domains of the MHC class II β chain or a portion or fragment or variant thereof (e.g., but not limited to, containing the MHC class II β1 domain or a portion or fragment or variant thereof, or the MHC class II β1 and β2 domains or a portion or fragment or variant thereof) can be linked (e.g., but not limited to, in-frame fusion or via a linker) to the Fos leucine zipper dimer motif. The linking (e.g., but not limited to, in-frame fusion or via a linker) can, for example, be at the C-terminus of the extracellular domains of the MHC class II α chain or a portion or fragment or variant thereof and at the C-terminus of the extracellular domains of the MHC class II β chain or a portion or fragment or variant thereof. Suitable adapters for linking Jun leucine zipper dimerization motifs and / or Fos leucine zipper dimerization motifs to MHC class II molecules are disclosed in more detail elsewhere herein. Optionally, in a non-limiting example, the adapter comprises SGGGGG (SEQ ID NO:1). Optionally, in a non-limiting example, the adapter consists essentially of SGGGGG (SEQ ID NO:1). Optionally, in a non-limiting example, the adapter consists of SGGGGG (SEQ ID NO:1).

[0095] In some embodiments, exemplary sequences of the Fos leucine zipper dimerization motif and the Jun leucine zipper dimerization motif are listed in SEQ ID NO:23 and 24, respectively.

[0096] As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed herein in some embodiments may comprise at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the sequence listed in SEQ ID NO:23. As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed herein in some embodiments may consist substantially of a sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence listed in SEQ ID NO:23. As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed herein in some embodiments may consist of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical sequences to those listed in SEQ ID NO:23. As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed herein in some embodiments may contain at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical sequences to those listed in SEQ ID NO:23. As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed herein in some embodiments may consist substantially of a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence listed in SEQ ID NO:23. As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed herein in some embodiments may consist of a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence listed in SEQ ID NO:23.

[0097] Similarly, in some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein may comprise at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the sequence listed in SEQ ID NO:24. Likewise, in some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein may consist substantially of a sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence listed in SEQ ID NO:24. Similarly, in some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein may consist of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical sequences to those listed in SEQ ID NO: 24. Likewise, in some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein may comprise at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical sequences to those listed in SEQ ID NO: 24. Similarly, in some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein may consist substantially of a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence listed in SEQ ID NO:24.

[0098] In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein has between 90% and 100% identity with the sequence listed in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein has between 92% and 100% identity with the sequence listed in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein has between 94% and 100% identity with the sequence listed in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein has between 96% and 100% identity with the sequence listed in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein has between 98% and 100% identity with the sequence listed in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein has between 90% and 98% identity with the sequence listed in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein has between 90% and 96% identity with the sequence listed in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein has between 90% and 94% identity with the sequence listed in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein has between 90% and 92% identity with the sequence listed in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein has between 92% and 98% identity with the sequence listed in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein has between 94% and 96% identity with the sequence listed in SEQ ID NO:23.

[0099] In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein has between 90% and 100% identity with the sequence listed in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein has between 92% and 100% identity with the sequence listed in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein has between 94% and 100% identity with the sequence listed in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein has between 96% and 100% identity with the sequence listed in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein has between 98% and 100% identity with the sequence listed in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein has between 90% and 98% identity with the sequence listed in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein has between 90% and 96% identity with the sequence listed in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein has between 90% and 94% identity with the sequence listed in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein has between 90% and 92% identity with the sequence listed in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein has between 92% and 98% identity with the sequence listed in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein has between 94% and 96% identity with the sequence listed in SEQ ID NO:24.

[0100] In another exemplary embodiment, the MHC class II α chain or a portion or fragment or variant thereof and the MHC class II β chain or a portion or fragment or variant thereof are linked using an immunoglobulin scaffold (e.g., but not limited to an IgG scaffold). In one exemplary embodiment, the MHC class II α chain or a portion or fragment or variant thereof and the MHC class II β chain or a portion or fragment or variant thereof are linked to the immunoglobulin light chain variable region and the immunoglobulin heavy chain variable region, respectively, or vice versa. See, for example, Hamad et al. (1998), *Journal of Experimental Medicine* 188(9):1633-1640, which is incorporated herein by reference in its entirety for all purposes. In another exemplary embodiment, the hydrophobic transmembrane regions of the MHC class II α chain and the MHC class II β chain are replaced by the immunoglobulin light chain variable region and the immunoglobulin heavy chain variable region, respectively, or vice versa. In another exemplary embodiment, the extracellular domains of the MHC class II α chain or a portion or fragment or variant thereof (e.g., but not limited to, containing the MHC class II α1 domain or a portion or fragment or variant thereof, or the MHC class II α1 and α2 domains or a portion or fragment or variant thereof) may be linked (e.g., but not limited to, in-frame fusion or via adapters) to the immunoglobulin light chain variable region, and the extracellular domains of the MHC class II β chain or a portion or fragment or variant thereof (e.g., but not limited to, containing the MHC class II β1 domain or a portion or fragment or variant thereof, or the MHC class II β1 and β2 domains or a portion or fragment or variant thereof) may be linked (e.g., but not limited to, in-frame fusion or via adapters) to the immunoglobulin heavy chain variable region. In another exemplary embodiment, the extracellular domains of the MHC class II α chain or a portion or fragment or variant thereof (e.g., but not limited to, containing the MHC class II α1 domain or a portion or fragment or variant thereof, or the MHC class II α1 and α2 domains or a portion or fragment or variant thereof) may be linked (e.g., but not limited to, in-frame fusion or via a adapter) to the immunoglobulin heavy chain variable region, and the extracellular domains of the MHC class II β chain or a portion or fragment or variant thereof (e.g., but not limited to, containing the MHC class II β1 domain or a portion or fragment or variant thereof, or the MHC class II β1 and β2 domains or a portion or fragment or variant thereof) may be linked (e.g., but not limited to, in-frame fusion or via a adapter) to the immunoglobulin light chain variable region. The linking (e.g., but not limited to, in-frame fusion or via a adapter) may, for example, be at the C-terminus of the extracellular domains of the MHC class II α chain or a portion or fragment or variant thereof and at the C-terminus of the extracellular domains of the MHC class II β chain or a portion or fragment or variant thereof. Suitable adapters are disclosed in more detail elsewhere herein.

[0101] In some embodiments, the MHC class II α chain or a portion or fragment or variant thereof and / or the MHC class II β chain or a portion or fragment or variant thereof may be linked to a crystallizable (Fc) region or fragment of an immunoglobulin fragment (e.g., but not limited to, the IgG2a Fc domain, such as the mouse IgG2a Fc domain). See, for example, Arnold et al. (2002), *J. Immunol. Methods* 271(1-2):137-151 and Appel et al. (2000), *J. Biol. Chem.* 275(1):312-321, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the Fc region or fragment may contain Davisosome modifications (e.g., CH3 modifications that allow differential binding of Fc to protein A) to facilitate purification. See, for example, U.S. Patent No. 8,586,713, which is incorporated herein by reference in its entirety for all purposes. As a non-limiting example, the Fc segment used may include hinges, C H 2 and C H 3. Structural domains (e.g., but not limited to, where the MHC class II α chain or a portion or fragment or variant thereof, or the MHC class II β chain or a portion or fragment or variant thereof, replaces the F(ab) arm of the antibody). Hinge regions, for example, may augment the MHC class II α chain or a portion or fragment or variant thereof and / or the MHC class II β chain or a portion or fragment or variant thereof relative to the C-containing region. H 2 and C H 3. Mobility of the Fc segment of the domain. In one exemplary embodiment, the hydrophobic transmembrane region of the MHC class II α chain and / or the MHC class II β chain is replaced by an immunoglobulin Fc region or fragment. In one exemplary embodiment, the extracellular domain of the MHC class II α chain or a portion or fragment or variant thereof (e.g., but not limited to, containing the MHC class II α1 domain or a portion or fragment or variant thereof, or the MHC class II α1 and α2 domains or a portion or fragment or variant thereof) may be linked (e.g., but not limited to, in-frame fusion or via adapter) to the immunoglobulin Fc region or fragment, and / or the extracellular domain of the MHC class II β chain or a portion or fragment or variant thereof (e.g., but not limited to, containing the MHC class II β1 domain or a portion or fragment or variant thereof, or the MHC class II β1 and β2 domains or a portion or fragment or variant thereof) may be linked (e.g., but not limited to, in-frame fusion or via adapter) to the immunoglobulin Fc region or fragment. Connections (e.g., but not limited to, in-frame fusion or via adapters) may, for example, be at the C-terminus of the extracellular domain of the MHC class II α chain or a portion or fragment or variant thereof, and at the C-terminus of the extracellular domain of the MHC class II β chain or a portion or fragment or variant thereof. Suitable adapters are disclosed in more detail elsewhere herein.

[0102] Optionally, in some embodiments, the MHC class II α chain or a portion or fragment or variant thereof and / or the MHC class II β chain or a portion or fragment or variant thereof may be further linked to an immunoglobulin fragment crystallizable (Fc) region or a portion or fragment or variant thereof (e.g., but not limited to, allowing the formation of a divalent molecule). See, for example, Arnold et al. (2002) J. Immunol. Methods 271(1-2):137-151 and Appel et al. (2000) Journal of Biochemistry 275(1):312-321, each of which is incorporated herein by reference in its entirety for all purposes. As a non-limiting example, the Fc segment used may include hinges, C H 2 and C H 3. Domains. In one exemplary embodiment, the immunoglobulin Fc region or fragment may be linked (e.g., but not limited to, fused or via a linker) to the C-terminus of the Fos leucine zipper dimer motif and / or may be linked to the C-terminus of the Jun leucine zipper dimer motif. Suitable linkers are disclosed elsewhere herein.

[0103] In another exemplary embodiment, the MHC class II α chain, or a portion or fragment or variant thereof, and the MHC class II β chain, or a portion or fragment or variant thereof, are joined using a knob-pore strategy. A knob-pore is a strategy for heterodimerization in which the knob and pore variant are designed to perform heterodimerization by inserting the knob into a suitably designed pore on the partner chain or domain. See, for example, Ridgway et al. (1996), Protein Engineering 9(7):617-621, which is incorporated herein by reference in its entirety for all purposes. As a non-limiting example, the knob and pore may be designed in the Fc region or fragment of an immunoglobulin (e.g., C... H (3 domains). As another non-limiting example, the knob can be designed on the MHC class II α chain or a portion, fragment, or variant thereof to insert into a corresponding pore on the MHC class II β chain or a portion, fragment, or variant thereof, or vice versa. The knob is constructed by replacing an amino acid with a small side chain with an amino acid with a large side chain. In this case, a pore of the same or similar size as the knob can be produced by replacing an amino acid with a large side chain with an amino acid with a smaller side chain. As a non-limiting example, the knob can be constructed by replacing an amino acid with a small side chain with tyrosine or tryptophan, and the corresponding pore can be constructed by replacing an amino acid with a large side chain with alanine or threonine.

[0104] In another exemplary embodiment, the MHC class II α chain, or a portion, fragment, or variant thereof, and the MHC class II β chain, or a portion, fragment, or variant thereof, are linked based on a charge mutation. The contact residues between the MHC class II α chain, or a portion, fragment, or variant thereof, and the MHC class II β chain, or a portion, fragment, or variant thereof, can be charged amino acids or neutral amino acids. Charged amino acids are amino acid residues with charged side chains. These can be positively charged side chains, such as those present in arginine (Arg, R), histidine (His, H), and lysine (Lys, K), or negatively charged side chains, such as those present in aspartic acid (Asp, D) and glutamic acid (Glu, E). Neutral amino acids are any other amino acids without charged side chains. These neutral residues include serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (Glu, Q), cysteine ​​(Cys, C), glycine (Gly, G), proline (Pro, P), alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, T). As a non-limiting example, one or more positively charged amino acids can be engineered into the MHC class II α chain or a portion or fragment thereof or a variant thereof to interact with one or more negatively charged amino acids in the MHC class II β chain or a portion or fragment thereof or a variant thereof, or vice versa. As another non-limiting example, one or more negatively charged amino acids can be engineered into the MHC class II α chain or a portion, fragment, or variant thereof to interact with one or more positively charged amino acids in the MHC class II β chain or a portion, fragment, or variant thereof, or vice versa. As another non-limiting example, one or more negatively charged amino acids can be engineered into the MHC class II α chain or a portion, fragment, or variant thereof to interact with one or more positively charged amino acids engineered into the MHC class II β chain or a portion, fragment, or variant thereof, or vice versa. In some embodiments, charged amino acids can be engineered into the MHC class II α chain or a portion, fragment, or variant thereof, or into the MHC class II β chain or a portion, fragment, or variant thereof, by replacing neutral amino acid residues with charged amino acid residues. In some embodiments, charged amino acids can be engineered into the MHC class II α chain or a portion, fragment, or variant thereof, or the MHC class II β chain or a portion, fragment, or variant thereof, by replacing amino acid residues with charged amino acid residues (e.g., replacing negatively charged amino acid residues with positively charged amino acid residues, or replacing positively charged amino acid residues with negatively charged amino acid residues). In one embodiment, the MHC can be linked to the Fc region of an immunoglobulin containing a different charge mutation.See, for example, U.S. Patent No. 9,358,286, which is incorporated herein by reference in its entirety for all purposes.

[0105] In another embodiment, the MHC class II α chain, or a portion or fragment or variant thereof, and the MHC class II β chain, or a portion or fragment or variant thereof, are covalently linked (e.g., but not limited to, via a linker such as a peptide linker). See, for example, Burrows et al. (1999), Protein Engineering 12(9):771-778, which is incorporated herein by reference in its entirety for all purposes. Such MHC class II molecules may be single-chain MHC fusions. In one exemplary embodiment, a single-chain MHC fusion may be a minimal TCR-binding unit containing only α1 and β1 domains, or portions or fragments or variants thereof (or containing neither α2 nor β2 domains and neither transmembrane nor cytoplasmic domains of the α and β chains). In one exemplary embodiment, the hydrophobic transmembrane regions of the MHC class II α and β chains are replaced by linkers such as peptide linkers. In one exemplary embodiment, an extracellular domain of an MHC class II α chain or a portion or fragment or variant thereof (e.g., but not limited to, containing an MHC class II α1 domain or a portion or fragment or variant thereof, or MHC class II α1 and α2 domains or a portion or fragment or variant thereof) may be connected (e.g., but not limited to, in-frame fusion or via a linker) to an extracellular domain of an MHC class II β chain or a portion or fragment or variant thereof (e.g., but not limited to, containing an MHC class II β1 domain or a portion or fragment or variant thereof, or MHC class II β1 and β2 domains or a portion or fragment or variant thereof). Suitable linkers are described elsewhere herein. In one exemplary embodiment, the N-terminus of the α chain or a portion or fragment or variant thereof is connected to the C-terminus of the β chain or a portion or fragment or variant thereof. In some embodiments, the C-terminus of the α chain or a portion or fragment or variant thereof may be connected to the N-terminus of the β chain or a portion or fragment or variant thereof. Suitable linkers are disclosed in more detail elsewhere herein.

[0106] B. MHC ligand peptides and their linkage with MHC class II molecules

[0107] In some embodiments of the invention, the MHC ligand peptide in the peptide-MHC class II complex may comprise any peptide (i.e., an antigenic peptide) capable of binding MHC proteins in a manner that allows the MHC-peptide complex to bind to the T cell receptor (TCR) and achieve a T cell response. The characteristics of the antigenic peptide, such as length, amino acid composition, etc., may depend on several factors, including but not limited to the peptide's ability to bind within the peptide-binding groove, experimental conditions, and the antigen of interest. These factors can be determined using commercially available computer programs such as Protean II™ (Proteus) and SPOT™. The binding of the peptide to the MHC peptide-binding groove can control the spatial arrangement of MHC and / or peptide amino acid residues recognized by the TCR or pMHC-binding proteins produced by animals. This spatial control is partly due to hydrogen bonds formed between the peptide and the MHC protein. Based on knowledge of how peptides bind to various MHCs, the major MHC anchoring amino acids and surface-exposed amino acids that vary among different peptides can be determined. The binding of the peptide to the MHC class II molecule is stabilized by hydrophobic anchoring and hydrogen bond formation. The peptide employs a type II polyproline helix because it interacts with the binding groove. Unbound by theory, this conformation is believed to cause peptides to twist in a specific manner, with peptide side chains isolated in polymorphic pockets within MHC II proteins. See, for example, Ferrante and Gorski (2007), *Journal of Immunology* 178:7181-7189, which is incorporated herein by reference in its entirety for all purposes. Unbound by theory, these pockets are believed to typically accommodate the side chains of peptide residues at P1, P4, P6, and P9 sites and have been identified as primary anchor sites. In addition to these largely solvent-inaccessible interactions, sites with smaller pockets or frames within binding sites accommodating P2, P3, P7, and P10 residues are considered secondary or auxiliary anchor sites.

[0108] In some embodiments, non-limiting examples of MHC ligand peptides suitable for the disclosed peptide-MHC class II complex include peptides comprising p-3 to p-12 residues. In some embodiments, non-limiting examples of MHC ligand peptides suitable for the disclosed peptide-MHC class II complex include peptides consisting substantially of p-3 to p-12 residues. In some embodiments, non-limiting examples of MHC ligand peptides suitable for the disclosed peptide-MHC class II complex include peptides consisting of p-3 to p-12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising p-2 to p-11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting substantially of p-2 to p-11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of p-2 to p-11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising p-1 to p-10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of P-1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P1 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P1 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of P1 to P9 residues.

[0109] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-3 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-2 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-3 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-2 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-3 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-2 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-3 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-2 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-1 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P1 to P9 residues.

[0110] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-3 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-2 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-3 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-2 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-3 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-2 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-3 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-2 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-1 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P1 to P9 residues.

[0111] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-3 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-2 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-3 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-2 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-3 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-2 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-3 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-2 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-1 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P1 to P9 residues.

[0112] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-3 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-3 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-3 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-3 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-2 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-2 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-2 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-2 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P-1 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides containing P1 to P9 residues.

[0113] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-3 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-3 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-3 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-3 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-2 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-2 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-2 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-2 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P-1 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of P1 to P9 residues.

[0114] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-3 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-3 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-3 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-3 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-2 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-2 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-2 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-2 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P-1 to P9 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P1 to P12 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P1 to P11 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P1 to P10 residues. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides composed of P1 to P9 residues.

[0115] In one embodiment, the MHC ligand peptide comprises P1 to P12 residues. In one embodiment, the MHC ligand peptide is substantially composed of P1 to P12 residues. In one embodiment, the MHC ligand peptide is composed of P1 to P12 residues. In one embodiment, the MHC ligand peptide comprises P-1 to P11 residues. In one embodiment, the MHC ligand peptide is substantially composed of P-1 to P11 residues. In one embodiment, the MHC ligand peptide is composed of P-1 to P11 residues. In one embodiment, the MHC ligand peptide comprises P-1 to P9 residues. In one embodiment, the MHC ligand peptide is substantially composed of P-1 to P9 residues. In one embodiment, the MHC ligand peptide is composed of P-1 to P9 residues. In one embodiment, the MHC ligand peptide comprises P-3 to P9 residues. In one embodiment, the MHC ligand peptide is substantially composed of P-3 to P9 residues. In one embodiment, the MHC ligand peptide is composed of P-3 to P9 residues.

[0116] In some embodiments, non-limiting examples of antigenic peptides suitable for the disclosed peptide-MHC class II complex include peptides comprising an antigen or a portion or fragment or variant thereof selected from the group consisting of: autoantigens, tumor-associated antigens, infectious agents, toxins, allergens, or combinations thereof. In one exemplary embodiment, the MHC ligand peptide comprises at least a portion or fragment or variant of a human autoprotein associated with an autoimmune disease (e.g., but not limited to antigenic determinants). In another embodiment, the MHC ligand peptide comprises at least a portion or fragment or variant of a protein of an infectious agent (e.g., but not limited to bacteria, viruses, or parasites) (e.g., but not limited to antigenic determinants). In another embodiment, the MHC ligand peptide comprises at least a portion or fragment or variant of an allergen (e.g., but not limited to antigenic determinants). In yet another embodiment, the MHC ligand peptide comprises at least a portion or fragment or variant of a tumor-associated protein (e.g., but not limited to antigenic determinants). In another embodiment, the MHC ligand peptide is associated with T-cell-mediated diseases (e.g., but not limited to, T-cell-mediated autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, multiple sclerosis, celiac disease, Addison's disease, and hypothyroidism). In one embodiment, the MHC ligand peptide may be a gliadin peptide or a gliadin-derived peptide. In another embodiment, the MHC ligand peptide (e.g., a gliadin peptide or a gliadin-derived peptide) may comprise QLQPFPQPELPY (SEQ ID NO:44), PQPELPYPQPQL (SEQ ID NO:46), or FPQPEQPFPWQP (SEQ ID NO:45). In another embodiment, the MHC ligand peptide (e.g., gliadin peptide or gliadin-derived peptide) may consist substantially of QLQPFPQPELPY (SEQ ID NO:44), PQPELPYPQPQL (SEQ ID NO:46), or FPQPEQPFPWQP (SEQ ID NO:45). In yet another embodiment, the MHC ligand peptide (e.g., gliadin peptide or gliadin-derived peptide) may consist of QLQPFPQPELPY (SEQ ID NO:44), PQPELPYPQPQL (SEQ ID NO:46), or FPQPEQPFPWQP (SEQ ID NO:45).In another embodiment, the MHC ligand peptide (e.g., gliadin peptide or gliadin-derived peptide) may comprise QLQPFPQPELPY (SEQ ID NO:44), PQPELPYPQPQL (SEQ ID NO:46), FPQPEQPFPWQP (SEQ ID NO:45), QPFPQPELPYPQ (SEQ ID NO:69), QPFPQPEQPFPW (SEQ ID NO:70), QPFPQPELPY (SEQ ID NO:71), FPQPELPYPQ (SEQ ID NO:72), or FPQPEQPFPW (SEQ ID NO:73). In another embodiment, the MHC ligand peptide (e.g., gliadin peptide or gliadin-derived peptide) may consist essentially of QLQPFPQPELPY (SEQ ID NO:44), PQPELPYPQPQL (SEQ ID NO:46), FPQPEQPFPWQP (SEQ ID NO:45), QPFPQPELPYPQ (SEQ ID NO:69), QPFPQPEQPFPW (SEQ ID NO:70), QPFPQPELPY (SEQ ID NO:71), FPQPELPYPQ (SEQ ID NO:72), or FPQPEQPFPW (SEQ ID NO:73). In another embodiment, the MHC ligand peptide (e.g., gliadin peptide or gliadin-derived peptide) may be composed of QLQPFPQPELPY (SEQ ID NO:44), PQPELPYPQPQL (SEQ ID NO:46), FPQPEQPFPWQP (SEQ ID NO:45), QPFPQPELPYPQ (SEQ ID NO:69), QPFPQPEQPFPW (SEQ ID NO:70), QPFPQPELPY (SEQ ID NO:71), FPQPELPYPQ (SEQ ID NO:72), or FPQPEQPFPW (SEQ ID NO:73).

[0117] In some embodiments, the MHC ligand peptide can be of any suitable length for binding to MHC proteins in a manner that allows the MHC-peptide complex to bind to the TCR and elicit a T cell response. The length of the MHC ligand peptide can vary, for example, from about 5 to about 40 amino acids (e.g., but not limited to, about 6 to about 30 amino acids, about 8 to about 20 amino acids, about 10 to about 18 amino acids, about 12 to about 18 amino acids, about 13 to about 18 amino acids, about 9 to about 11 amino acids, or any size peptide between 5 and 40 amino acids, in integer increments (i.e., 5, 6, 7, 8, 9...40)). While naturally occurring MHC-II binding peptides vary from about 9 to about 40 amino acids, in almost all cases, the peptide can be truncated to a core of 9-11 amino acids without losing MHC binding activity or T cell recognition. In some embodiments, the MHC ligand peptide length can be about 5 to about 40 amino acids. In some embodiments, the MHC ligand peptide length can be about 10 to about 40 amino acids. In some embodiments, the MHC ligand peptide length can be from about 15 to about 40 amino acids. In some embodiments, the MHC ligand peptide length can be from about 20 to about 40 amino acids. In some embodiments, the MHC ligand peptide length can be from about 25 to about 40 amino acids. In some embodiments, the MHC ligand peptide length can be from about 30 to about 40 amino acids. In some embodiments, the MHC ligand peptide length can be from about 35 to about 40 amino acids. In some embodiments, the MHC ligand peptide length can be from about 5 to about 35 amino acids. In some embodiments, the MHC ligand peptide length can be from about 5 to about 30 amino acids. In some embodiments, the MHC ligand peptide length can be from about 5 to about 25 amino acids. In some embodiments, the MHC ligand peptide length can be from about 5 to about 20 amino acids. In some embodiments, the MHC ligand peptide length can be from about 5 to about 15 amino acids. In some embodiments, the MHC ligand peptide length can be from about 5 to about 10 amino acids. In some embodiments, the MHC ligand peptide length can be from about 10 to about 35 amino acids. In some embodiments, the MHC ligand peptide length can be from about 15 to about 30 amino acids. In some embodiments, the MHC ligand peptide length can be from about 20 to about 25 amino acids. In some embodiments, the MHC ligand peptide length can be from about 9 to about 11 amino acids. In some embodiments, the MHC ligand peptide length can be from about 10 to about 18 amino acids. In some embodiments, the MHC ligand peptide length can be from about 9 to about 15 amino acids. In some embodiments, the MHC ligand peptide length can be from about 9 to about 14 amino acids. In some embodiments, the MHC ligand peptide length can be from about 9 to about 13 amino acids. In some embodiments, the MHC ligand peptide length can be from about 9 to about 12 amino acids.In some embodiments, the MHC ligand peptide can be about 10 to about 15 amino acids. In some embodiments, the MHC ligand peptide can be about 10 to about 14 amino acids. In some embodiments, the MHC ligand peptide can be about 10 to about 13 amino acids. In some embodiments, the MHC ligand peptide can be about 10 to about 12 amino acids.

[0118] In some embodiments, the MHC ligand peptide can be of any suitable length for binding to MHC proteins in a manner that allows the MHC-peptide complex to bind to the TCR and elicit a T cell response. The length of the MHC ligand peptide can vary, for example, from 5 to 40 amino acids (e.g., but not limited to, 6 to 30 amino acids, 8 to 20 amino acids, 10 to 18 amino acids, 12 to 18 amino acids, 13 to 18 amino acids, 9 to 11 amino acids, or any size peptide between 5 and 40 amino acids, in integer increments (i.e., 5, 6, 7, 8, 9...40)). While native MHC-II binding peptides vary from 9 to 40 amino acids, in almost all cases, the peptide can be truncated to a core of 9-11 amino acids without losing MHC binding activity or T cell recognition. In some embodiments, the MHC ligand peptide length can be 5 to 40 amino acids. In some embodiments, the MHC ligand peptide length can be 10 to 40 amino acids. In some embodiments, the MHC ligand peptide length can be 15 to 40 amino acids. In some embodiments, the MHC ligand peptide length can be 20 to 40 amino acids. In some embodiments, the MHC ligand peptide length can be 25 to 40 amino acids. In some embodiments, the MHC ligand peptide length can be 30 to 40 amino acids. In some embodiments, the MHC ligand peptide length can be 35 to 40 amino acids. In some embodiments, the MHC ligand peptide length can be 5 to 35 amino acids. In some embodiments, the MHC ligand peptide length can be 5 to 30 amino acids. In some embodiments, the MHC ligand peptide length can be 5 to 25 amino acids. In some embodiments, the MHC ligand peptide length can be 5 to 20 amino acids. In some embodiments, the MHC ligand peptide length can be 5 to 15 amino acids. In some embodiments, the MHC ligand peptide length can be 5 to 10 amino acids. In some embodiments, the MHC ligand peptide length can be 10 to 35 amino acids. In some embodiments, the MHC ligand peptide length can be 15 to 30 amino acids. In some embodiments, the MHC ligand peptide length can be 20 to 25 amino acids. In some embodiments, the MHC ligand peptide length can be 9 to 11 amino acids. In some embodiments, the MHC ligand peptide length can be 10 to 18 amino acids. In some embodiments, the MHC ligand peptide length can be 9 to 15 amino acids. In some embodiments, the MHC ligand peptide length can be 9 to 14 amino acids. In some embodiments, the MHC ligand peptide length can be 9 to 13 amino acids. In some embodiments, the MHC ligand peptide length can be 9 to 12 amino acids. In some embodiments, the MHC ligand peptide length can be 10 to 15 amino acids. In some embodiments, the MHC ligand peptide length can be 10 to 14 amino acids.In some embodiments, the MHC ligand peptide can be 10 to 13 amino acids. In some embodiments, the MHC ligand peptide can be 10 to 12 amino acids.

[0119] (1) Connector

[0120] In some embodiments of the complex, at least one chain of an MHC class II molecule, or a portion, fragment, or variant thereof, and an MHC ligand peptide are associated to form a fusion protein. In one exemplary embodiment, the MHC class II molecule (e.g., but not limited to, an MHC class II β chain, or a portion, fragment, or variant thereof, or an MHC class II α chain, or a portion, fragment, or variant thereof) and the MHC ligand peptide can be linked by a linker (e.g., but not limited to, covalent linkage, such as via a peptide linker). As a non-limiting example, the MHC ligand peptide can be directly or indirectly linked to the N-terminus of the MHC class II β chain, or to the C-terminus of the MHC class II β chain, or to the N-terminus of the MHC class II α chain, or to the C-terminus of the MHC class II α chain, or to the C-terminus of the MHC class II α chain, or to the MHC class II α chain, or to the N-terminus of the MHC class II α chain, or to the MHC class II α chain, or to the N-terminus of the MHC class II β chain, or to ... As a non-limiting example, the peptide-MHC class II complex may comprise an MHC ligand peptide, a linker, and a portion, fragment, or variant of an MHC class II β chain from the amino terminus to the carboxyl terminus. As a non-limiting example, the linker may extend from the C-terminus of the MHC ligand peptide to the N-terminus of the MHC class II β chain, a portion, fragment, or variant thereof. The linker may be configured to allow the attached MHC ligand peptide to fold into the binding groove of the MHC class II molecule, thereby producing a functional peptide-MHC class II complex. The advantage of attaching peptides to MHC class II molecules via flexible linkers is that it ensures that the peptide occupies and remains associated with the MHC during biosynthesis, transport, and display.

[0121] The length of the linker connecting the MHC ligand peptide to the MHC class II molecule can be any suitable length. In one exemplary embodiment, the linker can be long enough for the MHC ligand peptide to reach and bind to the peptide-binding groove of the MHC class II molecule, and short enough that the linker does not substantially inhibit the binding between the MHC ligand peptide and the peptide-binding groove of the MHC class II molecule. Based on known structural information (e.g., but not limited to known tertiary structure information), the linker length can be designed to span a distance greater than the distance between the N-terminus and C-terminus being linked. The appropriate size and sequence of the linker can also be determined using conventional computer modeling techniques based on the predicted tertiary structure of the peptide-MHC class II complex. As a non-limiting example, according to the HLA-DQ structure stored as PDB code 1S9V, the length between the C-terminal C-α atom of the MHC ligand peptide and the N-terminal C-α atom of the MHC α subunit or MHC β subunit is approximately See, for example, Kim et al. (2004), Proceedings of the National Academy of Sciences 101(12):4175-4179, which is incorporated herein by reference in its entirety for all purposes. Therefore, if the C-terminus of an MHC ligand peptide is connected via a linker to the N-terminus of an MHC class II β chain or a portion or domain or fragment or variant thereof, or an MHC class II α chain or a portion or domain or fragment or variant thereof, then the length of the linker can be designed to exceed the distance between the C-terminus of the MHC ligand peptide and the N-terminus of the MHC class II α or β chain or a portion or domain or fragment or variant thereof when the MHC ligand peptide is located within the peptide-binding groove of an MHC class II molecule. For example, according to the measurements described above, it may be necessary to exceed… (each amino acid residue) A linker (containing one amino acid) is used to bridge the measured distance. Additional amino acids may also be included so that the linker can bypass the protein molecule surface between the two connection points.

[0122] As a non-limiting example, the length of the linker can be at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, or at least about 15 amino acids. Similarly, in some embodiments, the length of the linker can be no more than about 50 amino acids, no more than about 45 amino acids, no more than about 40 amino acids, no more than about 35 amino acids, no more than about 30 amino acids, no more than about 25 amino acids, no more than about 20 amino acids, or no more than about 15 amino acids. In some embodiments, the length of the linker can be between about 9 amino acids and about 50 amino acids. In some embodiments, the length of the linker can be between about 9 amino acids and about 45 amino acids. In some embodiments, the length of the linker can be between about 9 amino acids and about 40 amino acids. In some embodiments, the length of the linker can be between about 9 amino acids and about 35 amino acids. In some embodiments, the length of the linker can be between about 9 amino acids and about 30 amino acids. In some embodiments, the length of the linker can be between about 9 amino acids and about 25 amino acids. In some embodiments, the linker length can be between about 9 amino acids and about 20 amino acids. In some embodiments, the linker length can be between about 10 amino acids and about 45 amino acids. In some embodiments, the linker length can be between about 10 amino acids and about 40 amino acids. In some embodiments, the linker length can be between about 10 amino acids and about 35 amino acids. In some embodiments, the linker length can be between about 10 amino acids and about 30 amino acids. In some embodiments, the linker length can be between about 10 amino acids and about 25 amino acids. In some embodiments, the linker length can be between about 10 amino acids and about 20 amino acids. In some embodiments, the linker length can be between about 15 amino acids and about 45 amino acids. In some embodiments, the linker length can be between about 15 amino acids and about 40 amino acids. In some embodiments, the linker length can be between about 15 amino acids and about 35 amino acids. In some embodiments, the linker length can be between about 15 amino acids and about 30 amino acids. In some embodiments, the linker length can be between about 15 amino acids and about 25 amino acids. In some embodiments, the linker length can be between about 15 amino acids and about 20 amino acids. In some embodiments, the linker length can be between about 10 amino acids and about 50 amino acids. In some embodiments, the linker length can be between about 15 amino acids and about 50 amino acids. In some embodiments, the linker length can be between about 20 amino acids and about 50 amino acids.In some embodiments, the linker length can be between about 25 and about 50 amino acids. In some embodiments, the linker length can be between about 30 and about 50 amino acids. In some embodiments, the linker length can be between about 35 and about 50 amino acids. In some embodiments, the linker length can be between about 40 and about 50 amino acids. In some embodiments, the linker length can be between about 45 and about 50 amino acids. In some embodiments, the linker length can be between about 10 and about 20 amino acids. In some embodiments, the linker length can be between about 11 and about 19 amino acids. In some embodiments, the linker length can be between about 12 and about 18 amino acids. In some embodiments, the linker length can be between about 13 and about 17 amino acids. In some embodiments, the linker length can be between about 14 and about 16 amino acids. In some embodiments, the linker can be a peptide of any size with a length between 9 and 50 amino acids, in integer increments (i.e., 9, 10, 11, 12, 13...50). In one exemplary embodiment, the linker can be about 15 amino acids long.

[0123] As a non-limiting example, the length of the linker can be at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, or at least 15 amino acids. Similarly, in some embodiments, the length of the linker can be no more than 50 amino acids, no more than 45 amino acids, no more than 40 amino acids, no more than 35 amino acids, no more than 30 amino acids, no more than 25 amino acids, no more than 20 amino acids, or no more than 15 amino acids. In some embodiments, the length of the linker can be between 9 and 50 amino acids. In some embodiments, the length of the linker can be between 9 and 45 amino acids. In some embodiments, the length of the linker can be between 9 and 40 amino acids. In some embodiments, the length of the linker can be between 9 and 35 amino acids. In some embodiments, the length of the linker can be between 9 and 30 amino acids. In some embodiments, the length of the linker can be between 9 and 25 amino acids. In some embodiments, the length of the linker can be between 9 and 20 amino acids. In some embodiments, the linker length can be between 10 and 45 amino acids. In some embodiments, the linker length can be between 10 and 40 amino acids. In some embodiments, the linker length can be between 10 and 35 amino acids. In some embodiments, the linker length can be between 10 and 30 amino acids. In some embodiments, the linker length can be between 10 and 25 amino acids. In some embodiments, the linker length can be between 10 and 20 amino acids. In some embodiments, the linker length can be between 15 and 45 amino acids. In some embodiments, the linker length can be between 15 and 40 amino acids. In some embodiments, the linker length can be between 15 and 35 amino acids. In some embodiments, the linker length can be between 15 and 30 amino acids. In some embodiments, the linker length can be between 15 and 25 amino acids. In some embodiments, the linker length can be between 15 and 20 amino acids. In some embodiments, the linker length can be between 10 and 50 amino acids. In some embodiments, the linker length can be between 15 and 50 amino acids. In some embodiments, the linker length can be between 20 and 50 amino acids. In some embodiments, the linker length can be between 25 and 50 amino acids. In some embodiments, the linker length can be between 30 and 50 amino acids.In some embodiments, the linker length can be between 35 and 50 amino acids. In some embodiments, the linker length can be between 40 and 50 amino acids. In some embodiments, the linker length can be between 45 and 50 amino acids. In some embodiments, the linker length can be between 10 and 20 amino acids. In some embodiments, the linker length can be between 11 and 19 amino acids. In some embodiments, the linker length can be between 12 and 18 amino acids. In some embodiments, the linker length can be between 13 and 17 amino acids. In some embodiments, the linker length can be between 14 and 16 amino acids. In some embodiments, the linker can be a peptide of any size with a length between 9 and 50 amino acids, in integer increments (i.e., 9, 10, 11, 12, 13...50). In one exemplary embodiment, the linker length can be 15 amino acids.

[0124] Any suitable amino acid can be used as a linker. In some embodiments, non-limiting examples of suitable linkers, including flexible, rigid, and cleavable linkers, are reviewed, for example, in Chen et al. (2013), *Advanced Drug Delivery Review* 65(10):1357-1369, which is incorporated herein by reference in its entirety for all purposes. For amino acid sequences capable of spanning defined distances between protein segments or domains, the PDB database, such as the server at the Center for Integrative Bioinformatics, University of Amsterdam, can also be searched at ibi.vu.nl / programs / linkerdbwww.

[0125] The linkers used in the peptide-MHC class II complexes disclosed herein in some embodiments may have one or more of the following characteristics: the linker is flexible, the linker is non-immunogenic, the linker does not include charged amino acids, the linker includes polar amino acids, and any combination thereof. Flexibility allows the MHC ligand peptide to freely bind to and assemble into its native peptide-binding groove in the MHC class II molecule. For example, flexibility can be achieved by using a linker rich in small or hydrophilic amino acids (e.g., but not limited to glycine and serine). In some embodiments, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the amino acids in the linker may be glycine. In some embodiments, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the amino acids in the linker may be glycine. In some embodiments, the amino acids in the linker, between about 40% and about 80%, may be glycine. In some embodiments, the amino acids in the linker, between 40% and 80%, may be glycine. In some embodiments, the amino acids in the linker, between about 50% and about 80%, may be glycine. In some embodiments, the amino acids in the linker, between 50% and 80%, may be glycine. In some embodiments, the amino acids in the linker, between about 60% and about 80%, may be glycine. In some embodiments, the amino acids in the linker, between 60% and 80%, may be glycine. In some embodiments, the amino acids in the linker, between about 70% and about 80%, may be glycine. In some embodiments, the amino acids in the linker, between 70% and 80%, may be glycine. In some embodiments, the amino acids in the linker, between 70% and 80%, may be glycine. In some embodiments, the amino acids in the linker, between about 40% and about 70%, may be glycine. In some embodiments, the amino acids in the linker, between about 40% and about 60%, may be glycine. In some embodiments, the amino acids in the linker, between 40% and 60%, may be glycine. In some embodiments, the amino acids in the linker, between about 40% and about 50%, may be glycine. In some embodiments, the amino acids in the linker, between 40% and 50%, may be glycine. In some embodiments, the amino acids in the linker, between about 50% and about 70%, may be glycine. In some embodiments, the amino acids in the linker, between 50% and 70%, may be glycine. In some embodiments, the amino acids in the linker, between about 55% and about 65%, may be glycine. In some embodiments, the amino acids in the linker, between 55% and 65%, may be glycine. Including polar amino acids can help improve solubility.In some embodiments, non-limiting examples of polar amino acids include Arg, Asn, Asp, Glu, Gln, His, Lys, Ser, Thr, and Tyr. In one exemplary embodiment, one or more serines are included in the linker. Omitting charged amino acids (e.g., but not limited to Lys, Arg, Glu, and Asp) can help avoid electrostatic interactions with other amino acid side chains. In some embodiments of the peptide-MHC class II complexes disclosed herein, the linker is flexible and includes one or more polar amino acids. In some embodiments of the peptide-MHC class II complexes disclosed herein, the linker is flexible (e.g., but not limited to, including flexible amino acids such as Gly) and does not include any charged amino acids. In some embodiments of the peptide-MHC class II complexes disclosed herein, the linker is flexible and non-immunogenic. In some embodiments of the peptide-MHC class II complexes disclosed herein, the linker includes one or more polar amino acids and does not include any charged amino acids. In some embodiments of the peptide-MHC class II complexes disclosed herein, the linker is non-immunogenic and includes one or more polar amino acids. In some embodiments of the peptide-MHC class II complexes disclosed herein, the linker is non-immunogenic and does not contain any charged amino acids. In some embodiments of the peptide-MHC class II complexes disclosed herein, the linker is flexible, includes one or more polar amino acids, and does not contain any charged amino acids. In some embodiments of the peptide-MHC class II complexes disclosed herein, the linker is non-immunogenic, includes one or more polar amino acids, and does not contain any charged amino acids. In some embodiments of the peptide-MHC class II complexes disclosed herein, the linker is flexible, non-immunogenic, and includes one or more polar amino acids. In some embodiments of the peptide-MHC class II complexes disclosed herein, the linker is flexible, non-immunogenic, includes one or more polar amino acids, and does not contain any charged amino acids. In some embodiments of the peptide-MHC class II complexes disclosed herein, the linker is flexible, non-immunogenic, includes one or more polar amino acids, and does not contain any charged amino acids.

[0126] In some embodiments, the adapter suitable for the disclosed peptide-MHC class II complex is a cleavable adapter containing a cleavage site. As a non-limiting example, the adapter may contain the tobacco etch virus (TEV) protease cleavage site ENLYFQ (SEQ ID NO:22). However, other cleavage sites may also be used (e.g., but not limited to, thrombin-sensitive cleavage sites, furin-sensitive cleavage sites, rhinovirus 3C protease cleavage sites, or intestinal peptidase cleavage sites). See, for example, Waugh (2011) Protein Expression and Purification 80(2):283-293, which is incorporated herein by reference in its entirety for all purposes.

[0127] Some linkers suitable for the disclosed peptide-MHC class II complexes primarily comprise amino acids with small side chains, such as glycine, alanine, and serine (e.g., but not limited to glycine and serine). In some embodiments, exemplary linkers include glycine polymers (G). n Glycine-serine polymers (including, for example, (GS)) n (GSGGS) n (SEQ ID NO:2), (GGGS) n (SEQ ID NO:3) and (GGGGS) n (SEQ ID NO:4), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other well-known flexible connectors. (GGGGS) n The linker (SEQ ID NO:4) is particularly suitable because it comprises a flexible amino acid (Gly) and a polar amino acid (Ser) capable of forming hydrogen bonds, which improves solubility. Suitable linkers may include (GSGGS). n (SEQ ID NO:2), (GGGS) n (SEQ ID NO:3) and (GGGGS) n Any of (SEQ ID NO:4). Suitable connectors can be made primarily of (GSGGS). n (SEQ ID NO:2), (GGGS) n (SEQ ID NO:3) and (GGGGS) n Any one of (SEQ ID NO:4). A suitable connector may be made of (GSGGS). n (SEQ ID NO:2), (GGGS) n (SEQ ID NO:3) and (GGGGS) nAny of the components in (SEQ ID NO:4). In some embodiments, the peptide linker (e.g., a peptide linker for linking an MHC ligand peptide to an MHC class II molecule) may comprise about 2 to about 4 repeats of the sequence listed in SEQ ID NO:4. In some embodiments, the peptide linker (e.g., a peptide linker for linking an MHC ligand peptide to an MHC class II molecule) may comprise 2 to about 4 repeats of the sequence listed in SEQ ID NO:4, wherein one amino acid of one repeat is mutated to cysteine. In some embodiments, the peptide linker (e.g., a peptide linker for linking an MHC ligand peptide to an MHC class II molecule) may comprise 2 to about 4 repeats of the sequence listed in SEQ ID NO:4, wherein one amino acid of one repeat is mutated to cysteine. As a non-limiting example, cysteine ​​may be the first, second, third, or fourth amino acid of the linker (e.g., but not limited to, the second amino acid of the linker). Glycine and glycine-serine polymers can be used. Both glycine and serine are relatively unstructured and therefore can be used as neutral chain links between components. Glycine can even enter significantly more phi-psi spaces than alanine and is much less restricted than residues with longer side chains. In some embodiments, exemplary linkers may comprise the following amino acid sequences, including but not limited to GGSG (SEQ ID NO:5), GGSGG (SEQ ID NO:6), GGSG (SEQ ID NO:7), GGSGG (SEQ ID NO:8), GGGSG (SEQ ID NO:9), GSSSG (SEQ ID NO:10), SGGGGG (SEQ ID NO:11), GCGAGSGGGGSGGGGS (SEQ ID NO:12), GCGAGSGGGGSGGGGS (SEQ ID NO:13), GGGGSGGGGS (SEQ ID NO:14), GGGASGGGGSGGGGS (SEQ ID NO:15), GGGGSGGGGSGGGGS (SEQ ID NO:16), or GGGASGGGGS (SEQ ID NO:17), GGGGSGGGGSGGGGS (SEQ ID NO:18), GGGGSGGGGSGGGGSGGGS (SEQ ID NO:19), GCGGS (SEQ ID NO:19), GCGGS (SEQ ID NO:10), GGSG ... ID NO: 20), GCGGSGGGGSGGGGS (SEQ ID NO: 21), GGGGSENLYFQGGGGS (SEQ ID NO: 47), etc.In some embodiments, the exemplary linker may consist substantially of the following amino acid sequences, including but not limited to GGSG (SEQ ID NO:5), GGSGG (SEQ ID NO:6), GGSG (SEQ ID NO:7), GGSGG (SEQ ID NO:8), GGGSG (SEQ ID NO:9), GSSSG (SEQ ID NO:10), SGGGGG (SEQ ID NO:11), GCGASGGGGSGGGGS (SEQ ID NO:12), GCGASGGGGSGGGGS (SEQ ID NO:13), GGGGSGGGGS (SEQ ID NO:14), GGGASGGGGSGGGGS (SEQ ID NO:15), GGGGSGGGGSGGGGS (SEQ ID NO:16), or GGGASGGGGS (SEQ ID NO:17), GGGGSGGGGSGGGGS (SEQ ID NO:18), GGGGSGGGGSGGGGSGGGS (SEQ ID NO:18). NO: 19), GCGGS (SEQ ID NO: 20), GCGGSGGGGSGGGGS (SEQ ID NO: 21), GGGGSENLYFQGGGGS (SEQ ID NO: 47), etc. In some embodiments, exemplary linkers may consist of the following amino acid sequences, including but not limited to GGSG (SEQ ID NO:5), GGSGG (SEQ ID NO:6), GGSG (SEQ ID NO:7), GGSGG (SEQ ID NO:8), GGGSG (SEQ ID NO:9), GSSSG (SEQ ID NO:10), SGGGGG (SEQ ID NO:11), GCGASGGGGSGGGGS (SEQ ID NO:12), GCGASGGGGSGGGGS (SEQ ID NO:13), GGGGSGGGGS (SEQ ID NO:14), GGGASGGGGSGGGGS (SEQ ID NO:15), GGGGSGGGGSGGGGS (SEQ ID NO:16), or GGGASGGGGS (SEQ ID NO:17), GGGGSGGGGSGGGGS (SEQ ID NO:18), GGGGSGGGGSGGGGSGGGS (SEQ ID NO:18). NO: 19), GCGGS (SEQ ID NO: 20), GCGGSGGGGSGGGGS (SEQ ID NO: 21), GGGGSENLYFQGGGGS (SEQ ID NO: 47), etc.In one exemplary embodiment, the adapter (e.g., an adapter for linking an MHC ligand peptide to an MHC class II molecule) comprises GCGGSGGGGSGGGGS (SEQ ID NO: 21). In one exemplary embodiment, the adapter (e.g., an adapter for linking an MHC ligand peptide to an MHC class II molecule) is substantially composed of GCGGSGGGGSGGGGS (SEQ ID NO: 21). In one exemplary embodiment, the adapter (e.g., an adapter for linking an MHC ligand peptide to an MHC class II molecule) is composed of GCGGSGGGGSGGGGS (SEQ ID NO: 21).

[0128] In some embodiments of the linker, the linker polypeptide includes a cysteine ​​residue capable of forming a disulfide bond with a cysteine ​​residue present in an MHC class II molecule. In one exemplary embodiment, the linker may include a cysteine ​​residue capable of forming a disulfide bond with a cysteine ​​residue present in an MHC class II α chain or a portion or fragment or variant thereof, or an MHC class II β chain or a portion or fragment or variant thereof. As a non-limiting example, the cysteine ​​may be the first, second, third, or fourth amino acid of the linker (e.g., but not limited to, the second amino acid of the linker).

[0129] Although the above adapters are described for connecting MHC class II molecules (e.g., but not limited to, MHC class II β chains or portions or fragments or variants thereof, or MHC class II α chains or portions or fragments or variants thereof) and MHC ligand peptides, these adapters may also be used in any other contexts of adapter use described herein.

[0130] (2) Disulfide bridge

[0131] In some embodiments of the complex, an MHC ligand peptide or a linker connecting the MHC ligand peptide to an MHC class II molecule is attached to at least one chain, portion, fragment, or variant thereof of the MHC class II molecule via a disulfide bridge (i.e., a disulfide bond extending between a pair of oxidized cysteine ​​residues). In one exemplary embodiment, the MHC ligand peptide may contain a first cysteine ​​residue, or the linker may contain a first cysteine ​​residue, and the MHC class II molecule may contain a second cysteine ​​residue in the tertiary structure of the complex located proximal to the first cysteine ​​residue, such that a disulfide bridge is formed and the MHC ligand peptide is linked to the peptide-binding groove of the MHC class II molecule. Tertiary structure refers to the three-dimensional structure resulting from protein folding and covalent cross-linking. For example, proximity can be determined based on the available crystal structure. This disulfide bond can help position the MHC ligand peptide in the peptide-binding groove of the MHC class II molecule. Optionally, in some embodiments, the MHC ligand peptide may contain a first cysteine ​​residue and not other cysteine ​​residues, or the linker may contain a first cysteine ​​residue and not other cysteine ​​residues. Optionally, in some embodiments, if the MHC ligand peptide contains a first cysteine, the linker does not contain any other cysteine. Optionally, in some embodiments, if the linker contains a first cysteine, the MHC ligand peptide does not contain any other cysteine. Optionally, in some embodiments, the MHC class II molecule contains only a second cysteine ​​and does not contain any other cysteines or any other unpaired cysteines (e.g., no Cys capable of forming disulfide bonds within the MHC class II molecule). Optionally, in some embodiments, if the second cysteine ​​is present in the MHC class II α chain or a portion or fragment or variant thereof, the MHC class II α chain or a portion or fragment or variant thereof does not contain any other cysteines or any other unpaired cysteines (e.g., no Cys capable of forming disulfide bonds within the MHC class II molecule). Optionally, in some embodiments, if the second cysteine ​​is present in the MHC class II β chain or a portion, fragment, or variant thereof, the MHC class II β chain or a portion, fragment, or variant thereof does not contain any other cysteine ​​or any other unpaired cysteine ​​(e.g., no Cys capable of forming disulfide bonds within the MHC class II molecule). The cysteine ​​in the MHC class II molecule may be a naturally occurring cysteine ​​(i.e., present in an unmodified (i.e., wild-type) MHC class II molecule), or it may be a mutation (addition or substitution) relative to an unmodified (i.e., wild-type) MHC class II molecule. Similarly, in some embodiments, the cysteine ​​in the MHC ligand peptide may be a naturally occurring cysteine, or it may be a mutation (addition or substitution) in the MHC ligand peptide. If it is a mutation in the MHC ligand peptide, preferably it is opposite to the epitope formed by the peptide-MHC class II complex.The chain (or portion, fragment, or variant thereof) of the MHC class II molecule that is connected (i.e., covalently linked) to the linker and the chain (or portion, fragment, or variant thereof) of the MHC class II molecule that forms a disulfide bridge can be the same chain or different chains of the MHC class II molecule. In one exemplary embodiment, the linker can be connected to the β chain of the MHC class II molecule (i.e., the MHC class II β chain or a portion, fragment, or variant thereof), and the disulfide bridge can be formed between a cysteine ​​residue of the MHC ligand peptide or the linker and an α chain of the MHC class II molecule (i.e., the MHC class II α chain or a portion, fragment, or variant thereof). In some embodiments, the linker can be connected to the α chain of the MHC class II molecule (i.e., the MHC class II α chain or a portion, fragment, or variant thereof), and the disulfide bridge can be formed between a cysteine ​​residue of the MHC ligand peptide or the linker and a β chain of the MHC class II molecule (i.e., the MHC class II β chain or a portion, fragment, or variant thereof). In some embodiments, the linker may be attached to the α chain of an MHC class II molecule (i.e., the MHC class II α chain or a portion, fragment, or variant thereof), and a disulfide bridge may be formed between a cysteine ​​residue of the MHC ligand peptide or the linker and the α chain of the MHC class II molecule (i.e., the MHC class II α chain or a portion, fragment, or variant thereof). In some embodiments, the linker may be attached to the β chain of an MHC class II molecule (i.e., the MHC class II β chain or a portion, fragment, or variant thereof), and a disulfide bridge may be formed between a cysteine ​​residue of the MHC ligand peptide or the linker and the β chain of the MHC class II molecule (i.e., the MHC class II β chain or a portion, fragment, or variant thereof).

[0132] In some embodiments of the peptide-MHC class II complex, residues that are not cysteine ​​in the unmodified (i.e., wild-type) α chain (i.e., the MHC class II α chain or a portion, fragment, or variant thereof) or the unmodified (i.e., wild-type) β chain (i.e., the MHC class II β chain or a portion, fragment, or variant thereof) of the MHC class II molecule may be mutated to cysteine ​​based on their proximity in the tertiary structure of the complex to cysteine ​​in the MHC ligand peptide or in the linker connecting the MHC ligand peptide to the MHC class II molecule. In some embodiments of the peptide-MHC class II complex, cysteine ​​residues may be inserted into the MHC class II α chain or a portion, fragment, or variant thereof, or the MHC class II β chain or a portion, fragment, or variant thereof, based on their proximity in the tertiary structure of the complex to cysteine ​​in the MHC ligand peptide or in the linker connecting the MHC ligand peptide to the MHC class II molecule. In other words, the MHC class II molecule in the complex can be mutated relative to the corresponding wild-type MHC class II molecule to include a second cysteine ​​at a position proximal to the first cysteine ​​in the tertiary structure of the complex (i.e., in the MHC ligand peptide or linker). For example, the proximity can be determined based on the available crystal structure. In an exemplary embodiment, position 101 in the MHC class II α chain sequence listed in SEQ ID NO:49 or SEQ ID NO:55 or 56 can be mutated to cysteine ​​(R101C). Non-limiting examples of MHC class II α chain sequences mutated to cysteine ​​are listed in SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:57. In some embodiments, the position corresponding to position 101 in the MHC class II α chain sequence listed in SEQ ID NO:49 when the sequence of the subject MHC class II α chain or a portion or fragment or variant thereof is best aligned with SEQ ID NO:49 can be mutated to cysteine ​​(e.g., in the wild-type MHC class II α chain sequence listed in SEQ ID NO:49). Figure 3In the alignment of the full-length sequences of HLA-DPA1, HLA-DQA1, and HLA-DRA1, the position corresponding to the marker DQA1 R101 can be mutated to cysteine. For example, position 107 in SEQ ID NO:51, or the position corresponding to position 107 in SEQ ID NO:51 when the sequence of the subject MHC class II α chain or its part, fragment, or variant is best aligned with SEQ ID NO:51, can be mutated to cysteine. Similarly, position 101 in SEQ ID NO:52, or the position corresponding to position 101 in SEQ ID NO:52 when the sequence of the subject MHC class II α chain or its part, fragment, or variant is best aligned with SEQ ID NO:52, can be mutated to cysteine. For example, position 78 in SEQ ID NO:59, or the position corresponding to position 78 in SEQ ID NO:59 when the sequence of the subject MHC class II α chain, or its portion, fragment, or variant, is best aligned with SEQ ID NO:59, can be mutated to cysteine. Similarly, position 79 in SEQ ID NO:61, or the position corresponding to position 79 in SEQ ID NO:61 when the sequence of the subject MHC class II α chain, or its portion, fragment, or variant, is best aligned with SEQ ID NO:61, can be mutated to cysteine. Furthermore, position 76 in SEQ ID NO:62, or the position corresponding to position 76 in SEQ ID NO:62 when the sequence of the subject MHC class II α chain, or its portion, fragment, or variant, is best aligned with SEQ ID NO:62, can be mutated to cysteine. As another embodiment, position 79 of the MHC class II α chain sequence listed in SEQ ID NO:52 (HLA class II histocompatibility antigen, DRα chain; NCBI accession number P01903.1) can be mutated to cysteine ​​(F79C). A non-limiting example of an MHC class II α chain sequence with this cysteine ​​mutation is listed in SEQ ID NO:58. In some embodiments, the position corresponding to position 79 of the MHC class II α chain sequence listed in SEQ ID NO:52 when the sequence of the subject MHC class II α chain or a portion or fragment or variant thereof is best aligned with SEQ ID NO:52 can be mutated to cysteine ​​(e.g., in...). Figure 3In the alignment of the full-length sequences of HLA-DPA1, HLA-DQA1, and HLA-DRA1, the position corresponding to the marker DRA1 F79 can be mutated to cysteine. For example, position 79 in SEQ ID NO:49, or the position corresponding to position 79 in SEQ ID NO:49 when the sequence of the subject MHC class II α chain or its part, fragment, or variant is best aligned with SEQ ID NO:49, can be mutated to cysteine. Similarly, position 85 in SEQ ID NO:51, or the position corresponding to position 85 in SEQ ID NO:51 when the sequence of the subject MHC class II α chain or its part, fragment, or variant is best aligned with SEQ ID NO:51, can be mutated to cysteine. For example, position 56 in SEQ ID NO:59, or the position corresponding to position 56 in the subject MHC class II α chain or its portion, fragment, or variant, can be mutated to cysteine ​​when the sequence of the subject MHC class II α chain or its portion, fragment, or variant is best aligned with SEQ ID NO:59. Similarly, position 57 in SEQ ID NO:61, or the position corresponding to position 57 in SEQ ID NO:61, can be mutated to cysteine ​​when the sequence of the subject MHC class II α chain or its portion, fragment, or variant is best aligned with SEQ ID NO:61. Furthermore, position 54 in SEQ ID NO:62, or the position corresponding to position 54 in SEQ ID NO:62, can be mutated to cysteine ​​when the sequence of the subject MHC class II α chain or its portion, fragment, or variant is best aligned with SEQ ID NO:62.

[0133] In some embodiments of peptide-MHC class II complexes, cysteine ​​residues in the unmodified (i.e., wild-type) α chain (i.e., the MHC class II α chain or a portion, fragment, or variant thereof) or the unmodified (i.e., wild-type) β chain (i.e., the MHC class II β chain or a portion, fragment, or variant thereof) of the MHC class II molecule may be mutated to non-cysteine ​​residues to minimize disulfide scrambling (i.e., the formation of disulfide bonds between cysteine ​​residues other than those intended to be used). The amino acid that substituted for cysteine ​​may be selected to allow for proper folding of the MHC complex. This can be determined, for example, based on available crystal structures or by examining sequence alignments of closely related MHC sequences. In one exemplary embodiment, cysteine ​​is mutated to alanine because it has the smallest side chain and is therefore the least spatially disruptive. In one exemplary embodiment, the cysteine ​​residue at position 70 of the MHC class II α chain sequence listed in SEQ ID NO:49 (HLA class II histocompatibility antigen, DQα1 chain; NCBI accession number P01909.1) can be mutated. As a non-limiting example, it can be mutated to Ala, Trp, Arg, or Gln (based on sequence alignment, the unpaired Cys in DQA1*0501 is replaced by Trp, Arg, or Gln in the closest MHC sequence from another species). In one exemplary embodiment, the cysteine ​​residue at position 70 of the MHC class II α chain sequence listed in SEQ ID NO:49 or 53 can be mutated to alanine (C70A). Non-limiting examples of MHC class II α chain sequences mutated to alanine are listed in SEQ ID NO:56 and 54. In one exemplary embodiment, the cysteine ​​residue at position 70 of the MHC class II α chain sequence listed in SEQ ID NO:49 can be mutated to glutamine (C70Q). Non-limiting examples of MHC class II α-chain sequences where this position is mutated to glutamine are listed in SEQ ID NO:55 and 57. In some embodiments, the cysteine ​​residue corresponding to position 70 in the MHC class II α-chain sequence listed in SEQ ID NO:49 may be mutated (e.g., but not limited to, to alanine or glutamine) when the sequence of the subject MHC class II α-chain or its portion, fragment, or variant is best aligned with SEQ ID NO:49. Figure 3In the alignment of the full-length HLA-DPA1, HLA-DQA1, and HLA-DRA1 sequences, the position corresponding to the marker C70 of DQA1 can be mutated. For example, in the subject MHC class II α chain or its portion, fragment, or variant, when the sequence of the subject MHC class II α chain or its portion, fragment, or variant is best aligned with SEQ ID NO:51, the position corresponding to position 75 in SEQ ID NO:51 can be mutated. Similarly, in the subject MHC class II α chain or its portion, fragment, or variant, when the sequence of the subject MHC class II α chain or its portion, fragment, or variant is best aligned with SEQ ID NO:52, the position corresponding to position 69 in SEQ ID NO:52 can be mutated. For example, the cysteine ​​residue at position 47 in the MHC class II α chain sequence listed in SEQ ID NO:59, or the position corresponding to position 47 in SEQ ID NO:59 when the sequence of the subject MHC class II α chain or its portion, fragment, or variant is best aligned with SEQ ID NO:59, can be mutated. Similarly, the position corresponding to position 47 in SEQ ID NO:61 when the sequence of the subject MHC class II α chain or its portion, fragment, or variant is best aligned with SEQ ID NO:61, can be mutated. Furthermore, the position corresponding to position 44 in SEQ ID NO:62 when the sequence of the subject MHC class II α chain or its portion, fragment, or variant is best aligned with SEQ ID NO:62, can be mutated.

[0134] In one exemplary embodiment, the linker connecting the MHC ligand peptide to the MHC class II molecule may contain a first cysteine ​​residue, and the MHC class II molecule may contain a second cysteine ​​residue at a proximal position, such that a disulfide bridge is formed and the MHC ligand peptide is linked to the peptide binding groove of the MHC class II molecule. Optionally, in some embodiments, position 101 in the MHC class II α chain sequence listed in SEQ ID NO:49 may be mutated to a cysteine ​​residue (R101C), or the position in the sequence of the subject MHC class II α chain or a portion or fragment or variant thereof, when the sequence of the subject MHC class II α chain or a portion or fragment or variant thereof is best aligned with SEQ ID NO:49, corresponding to position 101 in the MHC class II α chain sequence listed in SEQ ID NO:49, may be mutated to a cysteine ​​residue. Optionally, in some embodiments, the cysteine ​​residue at position 70 of the MHC class II α chain sequence listed in SEQ ID NO:49 may be mutated (e.g., but not limited to, mutated to Ala, Trp, Arg, or Gln), or the cysteine ​​residue corresponding to position 70 of the MHC class II α chain sequence listed in SEQ ID NO:49 when the sequence of the subject MHC class II α chain or its portion, fragment, or variant is best aligned with SEQ ID NO:49 may be mutated (e.g., but not limited to, mutated to alanine or glutamine). Optionally, in some embodiments, the linker contains cysteine ​​residues in the first 3 or 4 residues, such as a first residue, a second residue, a third residue, or a fourth residue (and optionally, in some embodiments, no additional cysteine ​​residues are contained). In one exemplary embodiment, the linker may contain a cysteine ​​residue at position 3. In another exemplary embodiment, the linker may contain a cysteine ​​residue at position 2. Optionally, in some embodiments, the linker comprises 15 amino acids (such as GCGGSGGGGSGGGGS (SEQ ID NO:21)) including a Cys disulfide bond attached to position 101 of the MHC class II α chain (or the position in the sequence of the subject MHC class II α chain or a portion or fragment or variant thereof that corresponds to position 101 in the MHC class II α chain sequence listed in SEQ ID NO:49 when the subject MHC class II α chain or a portion or fragment or variant thereof is best aligned with SEQ ID NO:49).Optionally, in some embodiments, the linker consists essentially of 15 amino acids (such as GCGGSGGGGSGGGGS (SEQ ID NO:21)) including a Cys disulfide bond attached to position 101 of the MHC class II α chain (or the position in the sequence of the subject MHC class II α chain or a portion or fragment or variant thereof that corresponds to position 101 in the MHC class II α chain sequence listed in SEQ ID NO:49 when the subject MHC class II α chain or a portion or fragment or variant thereof is best aligned with SEQ ID NO:49). Optionally, in some embodiments, the linker consists of 15 amino acids (such as GCGGSGGGGSGGGGS (SEQ ID NO:21)) including a Cys bond attached to a disulfide bond at position 101 of the MHC class II α chain (or the position in the sequence of the subject MHC class II α chain or a portion or fragment or variant thereof that corresponds to position 101 in the MHC class II α chain sequence listed in SEQ ID NO:49 when best aligned with SEQ ID NO:49). Optionally, in some embodiments, the MHC class II molecule is an HLA-DQ MHC class II molecule (e.g., but not limited to HLA-DQ2), an HLA-DR MHC class II molecule (e.g., but not limited to HLA-DR2), or an HLA-DP MHC class II molecule.

[0135] In another embodiment, the MHC ligand peptide may contain a first cysteine ​​residue, and the MHC class II molecule may contain a second cysteine ​​residue at a proximal position, such that a disulfide bridge is formed, linking the MHC ligand peptide to the peptide binding groove of the MHC class II molecule. In one exemplary embodiment, the P1 anchoring site of the MHC ligand peptide may be a cysteine ​​residue. In another embodiment, the P4 anchoring site of the MHC ligand peptide may be a cysteine ​​residue. In another embodiment, the P6 anchoring site of the MHC ligand peptide may be a cysteine ​​residue. In yet another embodiment, the P9 anchoring site of the MHC ligand peptide may be a cysteine ​​residue. Optionally, in some embodiments, the cysteine ​​residue at position 70 of the MHC class II α chain sequence listed in SEQ ID NO:49 may be mutated (e.g., but not limited to, mutated to Ala, Trp, Arg, or Gln), or the cysteine ​​residue corresponding to position 70 of the MHC class II α chain sequence listed in SEQ ID NO:49 when the sequence of the subject MHC class II α chain or its portion or fragment or variant is best aligned with SEQ ID NO:49 may be mutated (e.g., but not limited to, mutated to alanine or glutamine). Optionally, in some embodiments, the MHC ligand peptide contains cysteine ​​residues in the first 3 or 4 residues, such as the first residue, second residue, third residue, or fourth residue (and optionally, in some embodiments, no additional cysteine ​​residues are contained). Optionally, in some embodiments, the linker comprises 15 amino acids (such as GCGGSGGGGSGGGGS (SEQ ID NO:21)) including a Cys disulfide bond attached to position 101 of the MHC class II α chain (or the position in the sequence of the subject MHC class II α chain or a portion or fragment or variant thereof that corresponds to position 101 in the MHC class II α chain sequence listed in SEQ ID NO:49 when the subject MHC class II α chain or a portion or fragment or variant thereof is best aligned with SEQ ID NO:49). Optionally, in some embodiments, the linker consists essentially of 15 amino acids (such as GCGGSGGGGSGGGGS (SEQ ID NO:21)) including a Cys disulfide bond attached to position 101 of the MHC class II α chain (or the position in the sequence of the subject MHC class II α chain or a portion or fragment or variant thereof that corresponds to position 101 in the MHC class II α chain sequence listed in SEQ ID NO:49 when the subject MHC class II α chain or a portion or fragment or variant thereof is best aligned with SEQ ID NO:49).Optionally, in some embodiments, the linker consists of 15 amino acids (such as GCGGSGGGGSGGGGS (SEQ ID NO:21)) including a Cys bond attached to a disulfide bond at position 101 of the MHC class II α chain (or the position in the sequence of the subject MHC class II α chain or a portion or fragment or variant thereof that corresponds to position 101 in the MHC class II α chain sequence listed in SEQ ID NO:49 when best aligned with SEQ ID NO:49). Optionally, in some embodiments, the MHC class II molecule is an HLA-DQ MHC class II molecule (e.g., but not limited to HLA-DQ2), an HLA-DR MHC class II molecule (e.g., but not limited to HLA-DR2), or an HLA-DP MHC class II molecule.

[0136] C. Other components

[0137] In some embodiments of the invention, the composition comprising the peptide-MHC class II complex may further comprise other components. As a non-limiting example, the composition may also comprise one or more peptides or one or more other molecules capable of stimulating T helper cells, or may further comprise one or more immunostimulatory molecules capable of enhancing the immune response. Such T helper cell epitopes or immunostimulatory molecules may be linked (e.g., but not limited to, covalently linked) to the peptide-MHC class II complex, or they may be blended into the composition with the peptide-MHC class II complex rather than being physically linked to it. In one exemplary embodiment, such T helper cell epitopes or immunostimulatory molecules may be linked (e.g., but not limited to, covalently linked) to the peptide-MHC class II complex (e.g., but not limited to, linked to the C-terminus of the peptide-MHC class II complex). As a non-limiting example, T helper cell epitopes or immunostimulatory molecules may be indirectly or directly linked to the C-terminus of an MHC class II molecule (e.g., but not limited to, a portion or fragment or variant of the MHC class II α chain and / or a portion or fragment or variant of the MHC class II β chain). The covalent link may be direct or through a connector such as a peptide linker. In some embodiments, non-limiting examples of linkers suitable for the disclosed peptide-MHC class II complexes are described elsewhere herein.

[0138] As a non-limiting example of some embodiments, the T helper cell epitopes applicable to the disclosed peptide-MHC class II complex are pan-DR binding epitopes (PADRE) peptides or molecules designed based on their binding activity to most HLA-DR (human MHC class II) molecules. PADRE stands for "pan-DR binding epitope," and it is used to design T helper cell epitopes based on mouse MHC class II molecules. bThe mouse MHC-II binding sequence, which presents a “universal” MHC-II epitope in haplotypes to enhance the immune response, is described in Alexander et al. (2000) *Journal of Immunology* 164(3):1625-1633, which is incorporated herein by reference in its entirety for all purposes. It can be fused to the end of an antigen used in immunization, and its uptake by antigen-presenting cells and presentation of MHC-II improve the overall immune response. See, for example, US 6,413,935; US 5,736,142; and Alexander et al. (1994) *Immunity* 1(9):751-761, each of which is incorporated herein by reference in its entirety for all purposes. These peptides have been shown to contribute to generating a variety of immune responses against antigens. In some embodiments, the PADRE peptide may comprise AKFVAAWTLKAAA (SEQ ID NO:25). In some embodiments, the PADRE peptide may consist essentially of AKFVAAWTLKAAA (SEQ ID NO:25). In some implementations, the PADRE peptide may consist of AKFVAAWTLKAAA (SEQ ID NO:25).

[0139] Similar to PADRE, peptides derived from lymphocytic choriomeningovirus (LCMV) (e.g., but not limited to, LCMV glycoprotein (GP), nucleoprotein (NP), or zinc-binding protein (Z)) are alternative small peptides that can be used to enhance the immune response by binding to MHC-II. In some embodiments, such peptides may be used in addition to or as an alternative to PADRE. In some embodiments, the LCMV peptide used may be an LCMV-specific MHC class II restricted CD4+ T cell epitope. In some embodiments, the LCMV peptide used may comprise one or more of the following sequences: TMFEALPHIIDEVIN (epitope GP) 6-20 ;SEQ ID NO:26);GIKAVYNFATCGIFA(epistole GP 31-45 ;SEQ ID NO:27);DIYKGVYQFKSVEFD(epitaxy GP 66-80 ;SEQ ID NO:28);TSAFNKKTFDHTLMS(epitope GP 126-140 ;SEQID NO:29);DAQSAQSQCRTFRGR(epitaxy GP 176-190 ;SEQ ID NO:30);TFRGRVLDMFRTAFG(epitope GP 186-200 ;SEQ ID NO:31);CDMLRLIDYNKAALS(epitope GP 316-330;SEQ ID NO:32); IEQEADNMITEMLRK (epistole GP 409-423 ;SEQ ID NO:33);EVKSFQWTQALRREL(epistole NP 6-20 ;SEQ ID NO:34);KNVLKVGRLSAEELM(epitope NP 86-100 ;SEQ ID NO:35); SERPQASGVYMGNLT (epitope NP 116-130 ;SEQID NO:36);PSLTMACMAKQSQTP(epitaxyNP 176-190 ;SEQ ID NO:37);EGWPYIACRTSIVGR(epitope NP 311-325 ;(SEQ ID NO:38);SQNRKDIKLIDVEMT(epitope NP 466-480 ;SEQ ID NO:39);GWLCKMHTGIVRDKK(epitope NP 496-510 ;SEQ ID NO:40); and SCKSCWQKFDSLVRC (epistole Z) 31-45 (SEQ ID NO: 41). In some embodiments, the LCMV peptide used may consist essentially of one or more of the following sequences: TMFEALPHIIDEVIN (epitope GP). 6-20 ;SEQ ID NO:26);GIKAVYNFATCGIFA (epistole GP 31-45 ;SEQ ID NO:27);DIYKGVYQFKSVEFD(epitaxy GP 66-80 ;SEQ ID NO:28);TSAFNKKTFDHTLMS(epitope GP 126-140 ;SEQID NO:29);DAQSAQSQCRTFRGR(epitaxy GP 176-190 ;SEQ ID NO:30);TFRGRVLDMFRTAFG(epitope GP 186-200 ;SEQ ID NO:31);CDMLRLIDYNKAALS(epitope GP 316-330 ;SEQ ID NO:32); IEQEADNMITEMLRK (epistole GP 409-423 ;SEQ ID NO:33);EVKSFQWTQALRREL(epistole NP 6-20 ;SEQ ID NO:34);KNVLKVGRLSAEELM(epitope NP 86-100 ;SEQ ID NO:35); SERPQASGVYMGNLT (epitope NP 116-130;SEQID NO:36);PSLTMACMAKQSQTP(epitaxyNP 176-190 ;SEQ ID NO:37);EGWPYIACRTSIVGR(epitope NP 311-325 ;(SEQ ID NO:38);SQNRKDIKLIDVEMT(epitope NP 466-480 ;SEQ ID NO:39);GWLCKMHTGIVRDKK(epitope NP 496-510 ;SEQ ID NO:40); and SCKSCWQKFDSLVRC (epistole Z) 31-45 (SEQ ID NO: 41). In some embodiments, the LCMV peptide used may consist of one or more of the following sequences: TMFEALPHIIDEVIN (epitope GP). 6-20 ;SEQ ID NO:26);GIKAVYNFATCGIFA(epistole GP 31-45 ;SEQ ID NO:27);DIYKGVYQFKSVEFD(epitaxy GP 66-80 ;SEQ ID NO:28);TSAFNKKTFDHTLMS(epitope GP 126-140 ;SEQID NO:29);DAQSAQSQCRTFRGR(epitaxy GP 176-190 ;SEQ ID NO:30);TFRGRVLDMFRTAFG(epitope GP 186-200 ;SEQ ID NO:31);CDMLRLIDYNKAALS(epitope GP 316-330 ;SEQ ID NO:32); IEQEADNMITEMLRK (epistole GP 409-423 ;SEQ ID NO:33);EVKSFQWTQALRREL(epistole NP 6-20 ;SEQ ID NO:34);KNVLKVGRLSAEELM(epitope NP 86-100 ;SEQ ID NO:35); SERPQASGVYMGNLT (epitope NP 116-130 ;SEQID NO:36);PSLTMACMAKQSQTP(epitaxyNP 176-190 ;SEQ ID NO:37);EGWPYIACRTSIVGR(epitope NP 311-325 ;(SEQ ID NO:38);SQNRKDIKLIDVEMT(epitope NP 466-480 ;SEQ ID NO:39);GWLCKMHTGIVRDKK(epitope NP 496-510;SEQ ID NO:40); and SCKSCWQKFDSLVRC (epistole Z) 31-45 (SEQ ID NO:41). See, for example, Botten et al. (2010) Microbiol.Mol.Biol.Rev. 74(2):157-170 and Mothe et al. (2007) Journal of Immunology 179(2):1058-1067, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the peptide may comprise SERPQASGVYMGNLT (SEQ ID NO:36). In some embodiments, the peptide may consist essentially of SERPQASGVYMGNLT (SEQ ID NO:36). In some embodiments, the peptide may consist of SERPQASGVYMGNLT (SEQ ID NO:36).

[0140] In some embodiments, a T-cell epitope (e.g., LCMV peptide) is added to the composition comprising a peptide-MHC class II complex. In some embodiments, multiple T-cell epitopes (e.g., but not limited to 2, 3, 4, 5, 6, 7, 8, 9, or 10 T-cell epitopes) are added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 1 to about 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 2 to about 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 3 to about 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 4 to about 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 5 to about 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 6 to about 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 7 to about 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 8 to about 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 9 to about 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 1 to about 9 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 1 to about 8 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 1 to about 7 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 1 to about 6 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 1 to about 5 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 1 to about 4 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 1 to about 3 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 1 to about 2 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex.In some embodiments, about 2 to about 9 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 3 to about 8 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 4 to about 7 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 4 to about 6 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, about 2 to about 4 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, each of the plurality of T-cell epitopes may be, but is not limited to, an LCMV peptide, wherein each LCMV peptide may comprise any of the sequences listed above. In some embodiments, each of the plurality of T-cell epitopes may be, but is not limited to, an LCMV peptide, wherein each LCMV peptide may consist substantially of any of the sequences listed above. In some embodiments, each of the plurality of T-cell epitopes may be, but is not limited to, an LCMV peptide, wherein each LCMV peptide may consist of any of the sequences listed above.

[0141] In some embodiments, a T-cell epitope (e.g., LCMV peptide) is added to the composition comprising a peptide-MHC class II complex. In some embodiments, multiple T-cell epitopes (e.g., but not limited to 2, 3, 4, 5, 6, 7, 8, 9, or 10 T-cell epitopes) are added to the composition comprising a peptide-MHC class II complex. In some embodiments, 1 to 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, 2 to 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, 3 to 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, 4 to 10 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, 5 to 10 T-cell epitopes may be added to the composition containing a peptide-MHC class II complex. In some embodiments, 6 to 10 T-cell epitopes may be added to the composition containing a peptide-MHC class II complex. In some embodiments, 7 to 10 T-cell epitopes may be added to the composition containing a peptide-MHC class II complex. In some embodiments, 8 to 10 T-cell epitopes may be added to the composition containing a peptide-MHC class II complex. In some embodiments, 9 to 10 T-cell epitopes may be added to the composition containing a peptide-MHC class II complex. In some embodiments, 1 to 9 T-cell epitopes may be added to the composition containing a peptide-MHC class II complex. In some embodiments, 1 to 8 T-cell epitopes may be added to the composition containing a peptide-MHC class II complex. In some embodiments, 1 to 7 T-cell epitopes may be added to the composition containing a peptide-MHC class II complex. In some embodiments, 1 to 6 T-cell epitopes may be added to the composition containing a peptide-MHC class II complex. In some embodiments, 1 to 5 T-cell epitopes may be added to the composition containing the peptide-MHC class II complex. In some embodiments, 1 to 4 T-cell epitopes may be added to the composition containing the peptide-MHC class II complex. In some embodiments, 1 to 3 T-cell epitopes may be added to the composition containing the peptide-MHC class II complex. In some embodiments, 1 to 2 T-cell epitopes may be added to the composition containing the peptide-MHC class II complex. In some embodiments, 2 to 9 T-cell epitopes may be added to the composition containing the peptide-MHC class II complex.In some embodiments, 3 to 8 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, 4 to 7 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, 4 to 6 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, 2 to 4 T-cell epitopes may be added to the composition comprising a peptide-MHC class II complex. In some embodiments, each of the plurality of T-cell epitopes may be, but is not limited to, an LCMV peptide, wherein each LCMV peptide may comprise any of the sequences listed above. In some embodiments, each of the plurality of T-cell epitopes may be, but is not limited to, an LCMV peptide, wherein each LCMV peptide may consist substantially of any of the sequences listed above. In some embodiments, each of the plurality of T-cell epitopes may be, but is not limited to, an LCMV peptide, wherein each LCMV peptide may consist of any of the sequences listed above.

[0142] In some embodiments, other peptides or molecules may also be used in the compositions provided herein to improve the immune response. Non-limiting examples include immunostimulants such as keyhole hemocyanin (KLH) or polypeptides that are cross-presented by multiple haplotypes by an immune host (e.g., but not limited to, mice or rats). Such peptides are molecules that can be, for example, fused to a peptide-MHC class II complex or provided alone (e.g., but not limited to being blended into the same composition).

[0143] In some embodiments, peptides or other tags may also be used in the composition to facilitate, for example, purification. In some embodiments, non-limiting examples of tags suitable for the disclosed peptide-MHC class II complex include, but are not limited to, E. coli biotin ligase (BirA), myc-myc-histidine (mmH), glutathione S-transferase (GST), maltose-binding protein (MBP), chitin-binding protein (CBP), FLAG, and 1D4 (i.e., the 1D4 epitope derived from the C-terminus of bovine rhodopsin). In some embodiments, the sequence of the BirA tag may comprise GLNDIFEAQKIEWHE (SEQ ID NO:42). In some embodiments, the sequence of the BirA tag may consist substantially of GLNDIFEAQKIEWHE (SEQ ID NO:42). In some embodiments, the sequence of the BirA tag may consist of GLNDIFEAQKIEWHE (SEQ ID NO:42). In some embodiments, the mmH tag sequence may contain EQKLISEEDLEQKLISEEDLHHHHHH (SEQ ID NO:43) or EQKLISEEDLGGEQKLISEEDLHHHHHH (SEQ ID NO:48). In some embodiments, the mmH tag sequence may consist substantially of EQKLISEEDLEQKLISEEDLHHHHHH (SEQ ID NO:43) or EQKLISEEDLGGEQKLISEEDLHHHHHH (SEQ ID NO:48). In some embodiments, the mmH tag sequence may consist of EQKLISEEDLEQKLISEEDLHHHHHH (SEQ ID NO:43) or EQKLISEEDLGGEQKLISEEDLHHHHH (SEQ ID NO:48).

[0144] III. Nucleic acids encoding peptide-MHC class II complexes

[0145] Nucleic acids encoding peptide-MHC class II complexes disclosed herein in some embodiments are also provided. Such nucleic acids may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or hybrids or derivatives of DNA or RNA. Optionally, in some embodiments, the nucleic acid encoding the peptide-MHC class II complex may be codon-optimized to efficiently translate into a protein in a particular cell or organism. As a non-limiting example, the nucleic acid encoding the peptide-MHC class II complex may be modified to replace codons that have a higher frequency of use in human cells, mammalian cells, rodent cells, mouse cells, rat cells, or any other host cell of interest compared to naturally occurring polynucleotide sequences. Any portion or fragment of the nucleic acid molecule may be generated by: (1) isolating the molecule from its natural environment; (2) using recombinant DNA techniques (e.g., but not limited to, PCR amplification or cloning); or (3) using chemical synthesis methods. The nucleic acid encoding the peptide-MHC class II complex may contain modifications to improve stability or reduce immunogenicity. Non-limiting examples of modification include: (1) alteration or substitution of one or two unlinked phosphate oxygens and / or one or more linked phosphate oxygens in the phosphodiester backbone; (2) alteration or substitution of ribose components, such as alteration or substitution of the 2' hydroxyl group on the ribose; (3) substitution of the phosphate moiety with a dephosphorylating linker; (4) modification or substitution of naturally occurring nucleobases; (5) substitution or modification of the ribose-phosphate backbone; (6) modification of the 3' or 5' end of oligonucleotides (e.g., but not limited to, removal, modification or substitution of the terminal phosphate group, or partial conjugation); and (7) modification of sugars.

[0146] In some embodiments, nucleic acids may be in the form of expression constructs as defined elsewhere herein. As a non-limiting example, nucleic acids may include regulatory regions (e.g., but not limited to, transcriptional or translational control regions), full-length or partial coding regions, and combinations thereof, that control the expression of nucleic acid molecules. As a non-limiting example, nucleic acids may be operatively linked to promoters that are active in the cells or organisms of interest. Promoters that may be used in such expression constructs include, for example, promoters active in one or more eukaryotic cells such as mammalian cells (e.g., non-human mammalian cells or human cells), or rodent cells such as (e.g., but not limited to, mouse cells or rat cells). Such promoters may be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters.

[0147] In some embodiments, nucleic acids may include functional equivalents of natural nucleic acid molecules encoding MHC molecules or peptides, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted and / or inverted in a manner that such modifications substantially do not interfere with the ability of the nucleic acid molecule to encode proteins capable of forming compositions comprising peptide-MHC class II complexes as described elsewhere herein (e.g., those that can be recognized by T cell receptors).

[0148] IV. Instructions for use of peptide-MHC class II complexes

[0149] A method for inducing an immune response in a subject is also provided, comprising administering to the subject an effective amount of a composition comprising a peptide-MHC class II complex as described elsewhere herein.

[0150] In some embodiments of the invention, the subject may include, for example, any type of animal or mammal. Mammals include, for example, humans, non-human mammals, non-human primates, monkeys, apes, cats, dogs, horses, bulls, deer, bison, sheep, rabbits, rodents (e.g., but not limited to, mice, rats, hamsters, and guinea pigs) and livestock (e.g., but not limited to, cattle breeds such as dairy cows and bulls; sheep breeds such as sheep and goats; and pig breeds such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domesticated animals and agricultural animals are also included. The term "non-human mammal" does not include humans. Specific non-limiting examples of non-human mammals include rodents such as mice and rats.

[0151] The term administration refers to the application of a composition to a subject or system (e.g., but not limited to cells, organs, tissues, organisms, or related components or collections of components). The route of administration may vary depending on, for example, the subject or system to which the composition is administered, the nature of the composition, the purpose of administration, etc. The term "administration" or "administering" is intended to include routes of introducing a peptide-MHC class II complex into a subject to perform its intended function (e.g., but not limited to, inducing or modulating an immune response). In some embodiments, non-limiting examples of routes of administration that may be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal), oral, inhalation, rectal, and percutaneous. As a non-limiting example, administration to a subject (e.g., but not limited to, humans or rodents) may be via bronchial (including bronchial infusion), buccal, intestinal, intradermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intravenous, intracardiac, mucosal, nasal, oral, rectal, subcutaneous, sublingual, local, tracheal (including intratracheal infusion), percutaneous, vaginal, and / or vitreous administration. Peptide-MHC class II complexes may be administered in tablet or capsule form (e.g., but not limited to, by injection, inhalation, ophthalmic lotion, ointment, suppository, etc.), topically via lotion or ointment, or rectally via suppository. Administration may be in the form of a bolus injection or may be performed by continuous infusion. Administration may involve intermittent or continuous administration (e.g., but not limited to, perfusion) for at least a selected period of time. Depending on the route of administration, peptide-MHC class II complexes may be coated or contained in selected materials to protect them from natural conditions that may adversely affect their ability to perform their intended function. Peptide-MHC class II complexes can be administered alone, or in combination with another agent (e.g., but not limited to immunostimulants), a pharmaceutically acceptable carrier, or both. Peptide-MHC class II complexes can be administered before, concurrently with, or after the administration of other agents. Furthermore, peptide-MHC class II complexes can also be administered in vivo in the form of their active metabolites or more active metabolites.

[0152] Methods for preparing antigen-binding proteins are also provided, comprising immunizing a non-human animal with a peptide-MHC class II complex as described elsewhere herein, allowing the non-human animal to generate an immune response to the peptide-MHC class II complex, and isolating cells (e.g., but not limited to, lymphocytes) or nucleic acids from the non-human animal, wherein the cells or nucleic acids contain or encode an antigen-binding protein that specifically binds to the peptide-MHC class II complex. As a non-limiting example, the antigen-binding domain may specifically bind (e.g., but not limited to, having an equilibrium dissociation constant (KD) in the micromolar, nanomolar, or picomolar range) to an epitope of the peptide-MHC class II complex. In some embodiments, the antigen-binding protein may be a therapeutic antigen-binding protein or an antibody (e.g., for a patient).

[0153] In one exemplary embodiment, the cells are B cells isolated from non-human animals, and the method further includes identifying nucleic acid sequences of immunoglobulin heavy and light chain variable regions encoding variable domains of the immunoglobulin heavy and light chains that specifically bind to the peptide-MHC class II complex upon pairing. Such methods may also include expressing the nucleic acid sequences in an expression system suitable for expressing antigen-binding proteins to form antigen-binding proteins comprising a dimer of the heavy and light chain variable domains that bind to the peptide-MHC class II complex.

[0154] In another embodiment, the method includes isolating nucleic acids from a non-human animal and obtaining immunoglobulin heavy chain variable region sequences and / or immunoglobulin light chain variable region sequences, respectively encoding antibodies that specifically bind to peptide-MHC class II complexes. Such methods may further include generating antibodies that bind to peptide-MHC class II complexes using the immunoglobulin heavy chain variable region sequences and / or immunoglobulin light chain variable region sequences.

[0155] In some embodiments of the method, cells (such as B cells) are recovered from non-human animals (e.g., but not limited to, from the spleen or lymph nodes). Cells can be fused with myeloma cell lines to prepare immortalized hybridoma cell lines, and such hybridoma cell lines can be screened and selected to identify hybridoma cell lines that produce antibodies containing hybrid heavy chains specific to antigens used for immunization.

[0156] In some embodiments of the method, immunization includes primary immunization of a nonhuman animal with a peptide-MHC class II complex (e.g., but not limited to administration thereto), resting the nonhuman animal for a period of time, and reimmunization of the nonhuman animal with a peptide-MHC class II complex (e.g., but not limited to, enhancing its immune response). In some embodiments of the method, the method includes concurrent immunization and / or boosting of the nonhuman animal with a helper T cell epitope, such as, but not limited to, pan-DR T helper epitopes (PADRE). See, for example, U.S. Patent No. 6,413,935 and Alexander et al. (1994) Immunization 1:751-61, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments of the method, the method includes primary immunization of a nonhuman animal with a peptide-MHC class II complex and boosting of the immunized animal with a peptide-MHC class II complex linked to a helper T cell epitope, such as, but not limited to, PADRE. In some embodiments of the method, the method includes primary immunization and boosting of the nonhuman animal with a peptide-MHC class II complex linked to a helper T cell epitope. In methods including priming and / or booster immunization with PADRE, the non-human animal can be a mouse with a C57 / BL6 genetic background. In some embodiments, the C57BL strain of mice can be selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10C, ​​and C57BL / Ola, or can be a hybrid of the aforementioned C57BL / 6 strain and another strain (e.g., but not limited to 129, BALB, etc.). In some embodiments of the method, the time period between the priming and booster immunization of the non-human animal is several days, at least one week, at least two weeks, at least three weeks, at least four weeks, or at least one month.

[0157] In some embodiments of the method, the non-human animal may contain human or humanized immunoglobulin heavy chain and / or light chain loci, enabling the non-human animal to provide human or humanized antigen-binding proteins containing human or humanized antigen-binding domains (e.g., but not limited to, human or humanized variable domains). Immunoglobulin loci containing human variable region gene segments are known in the art and can be seen as non-limiting examples in U.S. Patent Nos. 5,633,425; 5,770,429; 5,814,318; 6,075,181; 6,114,598; 6,150,584; 6,998,514; 7,795,494; and 7,910,798. 8,232,449;8,502,018;8,697,940;8,703,485;8,754,287;8,791,323;8,809,051;8,907,157;9,035,128;9,145,588;9,206,263;9,447,177;9,551,124;9,580,4 U.S. Patent Publications 91 and 9,475,559, each of which is incorporated herein by reference in its entirety for all purposes, and U.S. Patent Publications 20100146647, 20110195454, 20130167256, 20130219535, 20130326647, 20130096287 and 20150113668, each of which is incorporated herein by reference in its entirety for all purposes, and PCT Publications WO2007117410, WO2008151081, WO2009157771, WO2010039900, WO2011004192, WO2011123708 and WO2014093908, each of which is incorporated herein by reference in its entirety for all purposes. As a non-limiting example, a non-human animal may include unrearranged or rearranged human or humanized immunoglobulin heavy loci and / or unrearranged or rearranged human or humanized immunoglobulin light chain loci in its genome, enabling the non-human animal to provide human or humanized antigen-binding proteins containing human or humanized antigen-binding domains (e.g., but not limited to, human or humanized immunoglobulin variable domains). Optionally, in some embodiments, at least one of the human or humanized immunoglobulin heavy loci and / or human or humanized immunoglobulin light chain loci is unrearranged.

[0158] In some embodiments, such methods may further include cloning a nucleotide sequence encoding a human heavy chain or light chain variable region sequence (which may encode a histidine-modified human heavy chain variable domain and / or a histidine-modified human light chain variable domain, which may also be or independently a universal light chain variable domain) in a frame having a gene encoding a human heavy chain constant region (CH) or a light chain constant region (CL) to form a human binding protein sequence, and expressing the human binding protein sequence in suitable cells.

[0159] The method also provides for identifying T cells that are specific to antigenic peptides or peptide-MHC class II complexes, comprising immunizing a non-human animal with a peptide-MHC class II complex as described elsewhere herein, eliciting an immune response in the non-human animal to the peptide-MHC class II complex, and isolating T cells that respond to the peptide or peptide-MHC class II complex.

[0160] Methods for preparing nucleic acid sequences encoding TCR variable domains (e.g., TCRα and / or β variable domains) are also provided. In some embodiments, such methods may include immunizing a non-human animal with a peptide-MHC class II complex as described elsewhere herein, inducing an immune response in the non-human animal to the peptide-MHC class II complex, and thereby obtaining a nucleic acid sequence encoding a human TCR variable domain that binds to the peptide or peptide-MHC class II complex. In one embodiment, the method may further include preparing a nucleic acid sequence encoding a TCR variable domain operatively linked to a TCR constant region, which includes isolating T cells from the non-human animal described herein and obtaining therefrom a nucleic acid sequence encoding a TCR variable domain operatively linked to a TCR constant region. In some embodiments, the non-human animal may contain a humanized T cell receptor variable locus, and the method may include determining a nucleic acid sequence of a human TCR variable region expressed by T cells and cloning the human TCR variable region into a nucleotide construct containing a nucleic acid sequence of a human TCR constant region, such that the human TCR variable region is operatively linked to a human TCR constant region. Optionally, in some embodiments, the method may further include a human TCR expressed by the construct (e.g., in cells) that is specific to the peptide or peptide-MHC class II complex.

[0161] Methods for preparing T-cell receptors (TCRs) specific to antigenic peptides or peptide-MHC class II complexes are also provided, comprising immunizing a non-human animal with a peptide-MHC class II complex as described elsewhere herein, eliciting an immune response in the non-human animal to the peptide-MHC class II complex, and isolating T cells that respond to the peptide or peptide-MHC class II complex. In some embodiments, such methods may further include determining the nucleic acid sequence of a TCR variable region expressed by T cells, cloning the TCR variable region into a nucleotide construct containing the nucleic acid sequence of a TCR constant region, such that the TCR variable region is operatively linked to the TCR constant region, and optionally expressing (e.g., in cells) a TCR specific to the peptide or peptide-MHC class II complex by the construct. In some embodiments, the non-human animal may contain a humanized T-cell receptor variable locus, and the method may include determining the nucleic acid sequence of a human TCR variable region expressed by T cells and cloning the human TCR variable region into a nucleotide construct containing the nucleic acid sequence of a human TCR constant region, such that the human TCR variable region is operatively linked to the human TCR constant region. Optionally, in some embodiments, the method may further include a human TCR expressed by the construct (e.g., in cells) that is specific to the peptide or peptide-MHC class II complex.

[0162] In some embodiments, the identified T cells or TCRs specific to the antigenic peptide or peptide-MHC class II complex can be used for the treatment of the subject (e.g., adoptive T cell therapy). In some embodiments, such methods may include, for example, immunizing a non-human animal with a peptide-MHC class II complex as described elsewhere herein, inducing an immune response in the non-human animal to the peptide-MHC class II complex, isolating T cells that respond to the peptide or peptide-MHC class II complex (i.e., antigen-specific T cells), determining the nucleic acid sequence of the TCR expressed by the T cells, cloning the nucleic acid sequence of the TCR into an expression vector (e.g., a retroviral vector), introducing the vector into T cells derived from the subject, causing the T cells to express the antigen-specific T cell receptor, and infusing the T cells into the subject. In some embodiments, the antigen-specific T cell population is expanded prior to infusion into the subject. In some embodiments, the subject's immune cell population is immune-depleted prior to the infusion of the antigen-specific T cells.

[0163] In some embodiments of the method, the non-human animal may express a humanized T-cell receptor. See, for example, U.S. Patent No. 9,113,616, which is incorporated herein by reference in its entirety for all purposes. As a non-limiting example, in some embodiments of the method, the non-human animal may contain a humanized T-cell receptor variable locus. As a non-limiting example, the non-human animal may express humanized TCRα and β peptides (and / or humanized TCRδ and TCRγ peptides). In one embodiment, the non-human animal contains an unrearranged human TCR variable locus in its genome.

[0164] In some embodiments of the method, a non-human animal is made tolerant to at least one empty human or humanized MHC class II molecule, or at least its empty human peptide binding groove, but may generate an antigen-binding protein of the human (humanized) MHC molecule (e.g., but not limited to, an antigen-binding protein containing a human or humanized variable domain) when it is complexed with an antigenic (e.g., but not limited to, a heterologous) peptide. In some embodiments of the method, tolerance of a non-human animal to an empty human (humanized) MHC class II molecule is achieved by genetically modifying the non-human animal to include in its genome a nucleotide sequence encoding a human (humanized) MHC molecule or at least its human peptide binding groove, such that the non-human animal expresses the human (humanized) MHC molecule or at least its human peptide binding groove as an empty human (humanized) MHC molecule, or its empty human peptide binding groove. The same animal, genetically modified to contain nucleotides encoding human (humanized) MHC molecules, may be further modified to contain humanized immunoglobulin heavy and / or light chain loci expressing human or humanized antigen-binding proteins (e.g., but not limited to, antigen-binding proteins having human or humanized variable domains), and / or may be further modified to contain humanized T-cell receptor variable loci. In some embodiments of the method, the non-human animal contains nucleic acids encoding human or humanized MHC IIα polypeptides and / or human or humanized MHC IIβ polypeptides. See, for example, US 2019 / 0292263, which is incorporated herein by reference in its entirety for all purposes. The MHC II nucleotide sequence may encode a wholly human MHC II protein (e.g., but not limited to, human HLA class II molecules) or a partially human and partially non-human humanized MHC II protein (e.g., but not limited to, chimeric human / non-human MHC II proteins, e.g., containing chimeric human / non-human MHC IIα and β polypeptides). Genetically modified nonhuman animals whose genomes (e.g., but not limited to, at endogenous loci) contain nucleotide sequences encoding humanized (e.g., but not limited to, chimeric human / nonhuman) MHC II polypeptides are disclosed in U.S. Patent Nos. 8,847,005 and 9,043,996, each of which is incorporated herein by reference in its entirety for all purposes.

[0165] While tolerance to the human peptide-binding domain of chimeric human / non-human MHC molecules can be achieved through expression at an endogenous MHC locus, in some embodiments, such tolerance can also occur in non-human animals expressing human MHC class II molecules (or their functional peptide-binding domains) at ectopic loci. Furthermore, in some embodiments, when non-human animals are immunized with a human HLA molecule (or its peptide-binding domain and / or a derivative thereof) complexed with an antigenic peptide (e.g., but not limited to, a peptide heterologous to the non-human animal), non-human animals expressing empty human MHC class II molecules (or empty peptide-binding domains thereof) at ectopic loci and tolerating said molecules are capable of generating a specific immune response against human HLA molecules (or their peptide-binding domains or derivatives thereof) derived from the expressed human MHC class II molecules. Without being bound by theory, it is believed that tolerance in non-human animals occurs after expression of human or humanized MHC class II molecules. Therefore, human or humanized MHC class II molecules do not necessarily need to be expressed at an endogenous locus.

[0166] In some embodiments, such methods may also include disrupting tolerance to endogenous peptides. Immunization of non-human animals (e.g., but not limited to rodents, such as mice or rats) with antigenic peptide-MHC class II complexes to obtain specific peptide-MHC class II binding proteins and peptide-MHC class II specific T cells depends on the sequence differences between the endogenous protein and the presented heterologous protein in the non-human animal, enabling the non-human animal's immune system to recognize the peptide-MHC class II complex as non-self (i.e., exogenous). In some cases, generating antibodies and T cells / TCRs against peptide-MHC class II complexes that are highly homologous to self-peptide-MHC class II complexes can be a challenging task due to immune tolerance to self-peptide-MHC class II complexes. Methods for disrupting tolerance to self-peptides homologous to peptides of interest are well known. See, for example, U.S. Publication No. 20170332610, which is incorporated herein by reference in its entirety for all purposes. In some implementations of such methods, methods for disrupting tolerance to endogenous peptides include modifying the non-human animals described herein to include the deletion (e.g., but not limited to, knockout mutations) of an autopeptide that is highly homologous to the peptide of interest.

[0167] The non-human animals used in the methods disclosed in some embodiments herein may include, for example, any type of non-human animal, such as mammals. Mammals include, for example, humans, non-human mammals, non-human primates, monkeys, apes, cats, dogs, horses, bulls, deer, bison, sheep, rabbits, rodents (e.g., but not limited to, mice, rats, hamsters, and guinea pigs) and livestock (e.g., but not limited to, cattle breeds such as dairy cows and bulls; sheep breeds such as sheep and goats; and pig breeds such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domesticated animals and agricultural animals are also included. The term "non-human mammal" does not include humans. Specific non-limiting examples of non-human mammals include rodents such as mice and rats.

[0168] For all purposes, all patent applications, websites, other publications, registration numbers, etc., cited above or below are incorporated herein by reference in their entirety as if each individual item were individually and specifically indicated to be incorporated by reference. Unless otherwise specifically stated, any feature, step, element, embodiment, or aspect of the invention may be used in combination with any other feature, step, element, embodiment, or aspect. Although the invention has been described in detail by way of illustration and example for clarity and understanding, it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

[0169] Brief description of the sequence

[0170] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations of nucleotide bases and three-letter codes for amino acids. Nucleotide sequences follow the standard convention of starting at the 5' end of the sequence and proceeding forward (i.e., from left to right in each row) to the 3' end. Only one strand is shown for each nucleotide sequence, but any mention of a shown strand should be understood to include the complementary strand. When a nucleotide sequence encoding an amino acid sequence is provided, it should be understood that a degenerate codon variant encoding the same amino acid sequence is also provided. When a DNA sequence encoding an amino acid sequence is provided, it should be understood that an RNA sequence encoding the same amino acid sequence (by replacing thymine with uracil) is also provided. Amino acid sequences follow the standard convention of starting at the N-terminus of the sequence and proceeding forward (i.e., from left to right in each row) to the C-terminus.

[0171] Table 1. Sequence description.

[0172]

[0173]

[0174]

[0175]

[0176] Example

[0177] Example 1. Design of peptide-MHC II protein constructs

[0178] Examples of soluble peptide-MHC I protein constructs have been previously described. Such constructs can be used for a variety of applications, including those for… Rodents are immunized to produce antibodies against in-groove peptides. This example describes the design of peptide-MHC II protein constructs in which the α and β chains of an MHC II molecule are anchored together and anchored to a peptide in its groove. These can be used for a variety of applications, such as generating soluble MHC II constructs to act as immunogens, and generating membrane-anchored MHC II proteins for other applications, including recruiting T cells expressing MHC II class-peptide-specific T cell receptors (TCRs). Soluble or membrane-anchored MHC II proteins can also be used to specifically target T cells expressing MHC II class-peptide-specific T cell receptors (TCRs) to modulate T cell activity or viability in different disease settings.

[0179] like Figure 1 As shown, several soluble peptide-MHC II constructs were designed. Descriptions of the soluble peptide-MHC II constructs are provided in Table 2. Some constructs include the *E. coli* biotin ligase (BirA) and myc-myc-histidine (mmH) tags, but other tags (e.g., but not limited to glutathione S-transferase (GST), maltose-binding protein (MBP), chitin-binding protein (CBP), FLAG, or 1D4) can also be used. Alignments of the full-length DQ2α chain segment used in the constructs (containing C70Q mutations or R101C and C70A mutations) are shown in [Table 2]. Figure 2The sequence is shown in the diagram. For the C70 mutation, the numbering may vary based on a reference sequence or a selected signal sequence for a given construct. C70 is the position in the full-length HLA-II DQα1 strand sequence designated as UniProt accession number P01909-1 (SEQ ID NO:49). The full-length HLA-II DQα1 strand sequence with the C70Q mutation is listed in SEQ ID NO:55, and the full-length HLA-II DQα1 strand sequence with the R101C and C70A mutations is listed in SEQ ID NO:54. The portion of the full-length HLA-II DQα1 strand contained in the soluble HLA-DQ2 construct tested below includes residues 24-216 of SEQ ID NO:49 (without R101 or C70 mutation), SEQ ID NO:55 (C70Q mutation), or SEQ ID NO:54 (R101C and C70A mutations). The full-length HLA-II DQβ1 chain sequence is designated as NCBI accession number NP_001230891.1 (SEQ ID NO:50). The portion of the full-length HLA-II DQβ1 chain contained in the soluble HLA-DQ2 construct for the following tests includes residues 33-230 of SEQ ID NO:50. Alignment of the full-length α-chain segments from different HLA class II alleles is shown in [the provided text]. Figure 3 As shown in the image.

[0180] Table 2. Soluble HLA-DQ2 constructs.

[0181]

[0182] Positive yields were observed using construct AC. The protein was purified using standard procedures including affinity and size exclusion chromatography. The final amount of purified protein was determined by UV absorbance and extinction coefficients calculated based on the protein's amino acid composition. Production yield was calculated by dividing the mass of the purified protein by the volume of the culture medium. Construct A yielded a purification yield of 14 mg / L when covalently linked to the QLQPFPQPELPY (SEQ ID NO:44, "QLQ" peptide). Construct C yielded a purification yield of 2.4 mg / L when covalently linked to the QLQ peptide (SEQ ID NO:44). Construct B was covalently linked alone to QLQ peptide (SEQ ID NO:44), FPQPEQPFPWQP (SEQ ID NO:45; "FPQ" peptide), and PQPELPYPQPQL (SEQ ID NO:46; "PQP" peptide), yielding purified concentrations of 204 mg / L, 36 mg / L, and 0.9 mg / L, respectively. The results are summarized in Table 3.

[0183] Measurable and consistent yields of soluble protein were achieved using a peptide-MHC II construct, the construct comprising:

[0184] (1) A jun / fos zipper at the C end that is connected to the α and β chains of MHC II via an SGGGGG (SEQ ID NO:1) connector;

[0185] (2) The introduced R101C mutation in the MHC IIα chain;

[0186] (3) A linker located at the N-terminus of the β-chain and connected to the peptide, wherein the linker includes an additional Cys mutation to allow disulfide bond formation between the linker Cys and the introduced R101C mutation in the MHC IIα chain; and

[0187] (4) Elimination of unpaired Cys in the α chain (C70A mutation).

[0188] Based on sequence alignment, the unpaired Cys in DQA1*0501 is replaced by Trp, Arg, or Gln in the closest MHC sequence from other species, so mutations of these residues can be used alternatively.

[0189] Table 3. Soluble HLA-DQ2 constructs - purification yield.

[0190]

[0191] The ability of MHC constructs to bind antibodies against MHC class II proteins was tested using two different Biacore assays. In both assays, the Octet HTX instrument was used, with Octet biosensors coated with either anti-mouse or anti-human Fc. Assays were run at 25°C with a run buffer of HBS-ET + 1 mg / mL BSA. The capture mixing rate and time were 1000 rpm and 1 minute, and the sample injection mixing rate and time were 1000 rpm and 2 minutes.

[0192] Construct C was analyzed to verify binding to the monoclonal antibody. In the first experiment, ~0.8 nm pan-class II anti-HLAmAb or anti-DR / DQ mAb was captured by immersing the anti-mFc-coated Octet biosensor in wells containing 100 nM mAb for 1 minute. The sensor capturing the mAb was then immersed in wells containing 200 nM construct C. Figure 4As shown in Table 4, soluble construct C binds to two anti-class II monoclonal antibodies captured on the surface of the anti-mFc sensor, but not to the isotype control mAb. In a second experiment, ~1 nm of construct C was captured by immersing the anti-hFc-coated Octet biosensor in wells containing 200 nM of construct C for 1 minute. The sensor containing the captured construct C was then immersed in wells containing 100 nM of pan-class II anti-HLA mAb or anti-DR / DQ mAb. Figure 5 As shown in Table 5, the soluble construct C captured on the surface of the anti-hFc sensor bound to both anti-class II monoclonal antibodies, but not to the isotype control mAb. The pan-class II anti-HLA antibody bound only to correctly folded HLA proteins, which allowed us to verify the conformational integrity of the generated and purified proteins.

[0193] Table 4. The binding of construct C to anti-class II mAbs trapped on the surface of the anti-mFc sensor.

[0194]

[0195] Table 5. The binding of construct C, which is captured on the surface of the anti-hFc sensor, to anti-class II mAb.

[0196] Pan-II anti-HLA mAb 1.02 0.40 Anti-DR / DQ mAb 0.94 0.40 Isotype control mAb 0.98 -0.01

[0197] Construct B was analyzed to verify binding to monoclonal antibodies. In the first experiment, ~0.8 nm of pan-class II anti-HLAmAb or anti-DR / DQ mAb was captured by immersing the anti-mFc-coated Octet biosensor in wells containing 100 nM mAb for 1 min. The sensor capturing the mAb was then immersed in wells containing 200 nM of construct B. Soluble construct B bound to both anti-class II monoclonal antibodies captured on the surface of the anti-mFc sensor, but not to the isotype control mAb. In the second experiment, ~1 nm of construct B was captured by immersing the anti-hFc-coated Octet biosensor in wells containing 200 nM of construct B for 1 min. The sensor capturing construct B was then immersed in wells containing 100 nM of pan-class II anti-HLAmAb or anti-DR / DQ mAb. Soluble construct B, captured on the surface of the anti-hFc sensor, bound to two anti-class II monoclonal antibodies but not to the isotype control mAb. The pan-class II anti-HLA antibody bound only to correctly folded HLA proteins, allowing us to verify the conformational integrity of the generated and purified proteins.

[0198] Example 2. Toleration in mice

[0199] Mice resistant to empty MHC class II molecules are generated or provided, wherein the MHC class II molecules are not derived from mice (e.g., but not limited to humans). For example, mice expressing MHC class II molecules from the corresponding endogenous locus or a locus other than the corresponding endogenous locus (e.g., but not limited to the ROSA26 locus) are first resistant to empty MHC class II molecules. These resistant mice are then injected with an immunogen (e.g., an MHC class II molecule containing an immunogenic peptide in a groove, such as construct A, construct B, or construct C from Example 1). Compared to mice not resistant to empty MHC class II molecules, these immunized mice produce specific antibody titers against this particular immunogen. Conversely, mice not resistant to and immunized with the subject MHC class II molecules produce antibodies that recognize not only the immunogenic peptide but also the MHC class II molecules. Thus, resistant mice immunized with the MHC class II molecules described herein are able to generate an antigen-specific immune response without producing antibodies against individual MHC class II molecules.

[0200] Example 3. Immunotolerance of mice using MHC protein constructs

[0201] Peptides tethered to HLA-DQB chains in the form of DNA and soluble dimer proteins were selected for immunization and screening. A schematic diagram of an example of a construct containing HLA-DQB chain-tethered peptides for immunization and screening is shown in [the diagram]. Figure 6A and 6B As shown in the image.

[0202] Mice (e.g., mice containing humanized immunoglobulin heavy chain and / or light chain variable region loci) are immunized with a peptide-MHC (pMHC) complex of interest, the complex comprising a peptide antigenic to the mouse and a human or humanized MHC class II molecule targeted for mouse tolerance. The mice are additionally and optionally boosted with the pMHC complex of interest, the booster also optionally linked to a helper T cell epitope. Antibodies (e.g., human or humanized antibodies expressed by humanized immunoglobulin heavy chain and / or light chain loci) are isolated from the immunized mice and their specificity for binding to the pMHC complex is tested.

[0203] Test mice were provided with nucleotide sequences for human MHC II molecular tolerance and containing the gene encoding a humanized immunoglobulin heavy chain locus (see, for example, Macdonald (2014) Proceedings of the National Academy of Sciences 111:5147-5152, which is incorporated herein by reference in its entirety for all purposes) and a humanized common light chain locus (see, for example, U.S. Patent Nos. 10,143,186; 10,130,081 and 9,969,814; U.S. Patent Publications Nos. 20120021409, 20120192300, 20130045492, 20130185821, 20130302836 and 20150313193, each of which is incorporated herein by reference in its entirety for all purposes). These test mice and non-tolerant control mice containing functional (e.g., mouse) ADAM6 genes (see, for example, U.S. Patent Nos. 8,642,835 and 8,697,940; each of which is incorporated herein by reference in its entirety for all purposes) and humanized immunoglobulin heavy and light chain loci were immunized with a pMHC complex containing heterologous in-groove peptides present in the background of HLA-DQ molecules, wherein the immunogen was administered either as a protein immunogen or as DNA encoding the pMHC complex. Mice were boosted at different time intervals via different routes using a pMHC complex immunogen with standard adjuvants or using a pMHC complex immunogen linked to a T-helper pan-DR epitope (PADRE) peptide. Pre-immunization serum was collected from mice prior to initiation of immunization. Blood was collected from mice periodically, and antiserum titers were determined on the corresponding antigens.

[0204] Antibody titers in serum against irrelevant antigens (i.e., antigens not observed in mice and therefore not expected to elicit a significant response at titration) and relevant antigens present in the HLA-DQ (in-slot peptide) background were determined using ELISA. Ninety-six-well microtiter plates (Thermo Scientific) were coated overnight with a tagged pMHC complex in phosphate-buffered saline (PBS, Irvine Scientific) containing either relevant in-slot peptides or irrelevant antigens present in the HLA-DQ background. The plates were washed with phosphate-buffered saline containing 0.05% Tween 20 (PBS-T, Sigma-Aldrich) and blocked with bovine serum albumin (BSA, Sigma-Aldrich) in PBS.

[0205] Pre-immune and antiserum were serially diluted in BSA-PBS and added to the plate. The plate was washed, and anti-mouse IgG-Fc-horseradish peroxidase (HRP) conjugated secondary antibody was added. The plate was washed and developed using 3,3',5,5'-tetramethylbenzidine (TMB) / H2O2 as substrate according to the manufacturer's recommended procedure, and the absorbance at 450 nm was recorded using a spectrophotometer (Victor, PerkinElmer). Antibody titers were calculated using Graphpad PRISM software. Antibody titers were calculated as interpolated serum dilution factors with a binding signal twice that of the background.

[0206] Compared with control mice that were not tolerant to human HLA class II molecules, tolerant mice to human HLA class II molecules or a portion thereof enhanced the mice’s ability to generate specific antibody responses to pMHCs of interest.

[0207] Example 4. Testing different peptides in the peptide-MHC II protein construct

[0208] Several soluble peptide-MHC II constructs with different gliadin immunogenic variants were designed to test different MHC ligand peptide parameters and confirm the expression of constructs with different ligand peptides. Specifically, variants of αI gliadin, αII gliadin, and ω2 gliadin were tested (Table 6).

[0209] Table 6. Glycol epitopes.

[0210] αI(SEQ ID NO:44) Q L Q P F P Q P E L P Y αII (SEQ ID NO:69) Q P F P Q P E L P Y P Q ω2(SEQ ID NO:70) Q P F P Q P E Q P F P W

[0211] The portion of the full-length HLA-II DQα1 chain contained in the soluble HLA-DQ2 constructs included in the following tests comprises residues 24-216 of SEQ ID NO:54 (R101C and C70A mutations; SEQ ID NO:64). The portion of the full-length HLA-II DQβ1 chain contained in the soluble HLA-DQ2 constructs included residues 33-230 of SEQ ID NO:50 (SEQ ID NO:60). Descriptions of the soluble peptide-MHC II constructs are provided in Table 7. Some constructs include PADRE, but other T-cell epitopes may also be used. As shown in Table 7, positive yields were observed using all constructs.

[0212] Table 7. Soluble HLA-DQ2 constructs.

[0213]

[0214]

[0215]

[0216] Proteins were purified using standard procedures including affinity and size exclusion chromatography. The amount of final protein obtained after purification was determined by UV absorbance and extinction coefficients calculated based on the protein's amino acid composition. Yields were calculated by dividing the mass of the purified protein by the volume of the culture medium.

[0217] The ability of peptide-MHC constructs to bind antibodies against MHC class II proteins was tested using two different Biacore assays. In both assays, the Octet HTX instrument was used, with Octet biosensors coated with either anti-mouse or anti-human Fc. Assays were run at 25°C with a run buffer of HBS-ET + 1 mg / mL BSA. The capture mixing rate and time were 1000 rpm and 1 minute, and the sample injection mixing rate and time were 1000 rpm and 2 minutes.

[0218] Each construct was analyzed to verify binding to monoclonal antibodies. In the first experiment, ~0.8 nm pan-class II anti-HLAmAb or anti-DR / DQ mAb was captured by immersing the anti-mFc-coated Octet biosensor in wells containing 100 nM mAb for 1 min. The sensor capturing the mAb was then immersed in wells containing 200 nM peptide-MHC construct. The soluble peptide-MHC construct bound to both anti-class II monoclonal antibodies captured on the surface of the anti-mFc sensor, but not to the isotype control mAb. In the second experiment, ~1 nm peptide-MHC construct was captured by immersing the anti-hFc-coated Octet biosensor in wells containing 200 nM peptide-MHC construct for 1 min. The sensor capturing the peptide-MHC construct was then immersed in wells containing 100 nM pan-class II anti-HLA mAb or anti-DR / DQ mAb. Soluble peptide-MHC constructs captured on the surface of the anti-hFc sensor bound to two anti-class II monoclonal antibodies, but not to the isotype control mAb. The pan-class II anti-HLA antibody bound only to correctly folded HLA proteins, which allowed us to verify the conformational integrity of the generated and purified proteins.

[0219] Next, mice tolerant to empty MHC class II molecules are generated or provided, wherein the MHC class II molecules are not derived from mice (e.g., but not limited to humans). For example, mice expressing MHC class II molecules from the corresponding endogenous locus or a locus other than the corresponding endogenous locus (e.g., but not limited to the ROSA26 locus) are first tolerant to empty MHC class II molecules. These tolerant mice are then injected with an immunogen (e.g., an MHC class II molecule containing an immunogenic peptide in the groove, such as any of the peptide-MHC constructs in Example 4). Compared to mice not tolerant to empty MHC class II molecules, these immunized mice produce specific antibody titers against this particular immunogen. Conversely, mice not tolerant and immunized with the subject MHC class II molecules produce antibodies that recognize not only the immunogenic peptide but also the MHC class II molecules. Thus, tolerant mice immunized with the MHC class II molecules described herein are able to generate an antigen-specific immune response without producing antibodies against individual MHC class II molecules.

[0220] Peptides tethered to HLA-DQB chains in the form of DNA and soluble dimer proteins are selected for immunization and screening, such as those described in Example 4.

[0221] Mice (e.g., mice containing humanized immunoglobulin heavy chain and / or light chain variable region loci) are immunized with a peptide-MHC (pMHC) complex of interest, the complex comprising a peptide antigenic to the mouse and a human or humanized MHC class II molecule targeted for mouse tolerance. The mice are additionally and optionally boosted with the pMHC complex of interest, the booster also optionally linked to a helper T cell epitope. Antibodies (e.g., human or humanized antibodies expressed by humanized immunoglobulin heavy chain and / or light chain loci) are isolated from the immunized mice and their specificity for binding to the pMHC complex is tested.

[0222] Test mice were provided with nucleotide sequences for human MHC II molecular tolerance and containing the gene encoding a humanized immunoglobulin heavy chain locus (see, for example, Macdonald (2014) Proceedings of the National Academy of Sciences 111:5147-5152, which is incorporated herein by reference in its entirety for all purposes) and a humanized common light chain locus (see, for example, U.S. Patent Nos. 10,143,186; 10,130,081 and 9,969,814; U.S. Patent Publications Nos. 20120021409, 20120192300, 20130045492, 20130185821, 20130302836 and 20150313193, each of which is incorporated herein by reference in its entirety for all purposes). These test mice and non-tolerant control mice containing functional (e.g., mouse) ADAM6 genes (see, for example, U.S. Patent Nos. 8,642,835 and 8,697,940; each of which is incorporated herein by reference in its entirety for all purposes) and humanized immunoglobulin heavy and light chain loci were immunized with a pMHC complex containing heterologous in-groove peptides present in the background of HLA-DQ molecules, wherein the immunogen was administered either as a protein immunogen or as DNA encoding the pMHC complex. Mice were boosted at different time intervals via different routes using a pMHC complex immunogen with standard adjuvants or using a pMHC complex immunogen linked to a T-helper pan-DR epitope (PADRE) peptide. Pre-immunization serum was collected from mice prior to initiation of immunization. Blood was collected from mice periodically, and antiserum titers were determined on the corresponding antigens.

[0223] Antibody titers in serum against irrelevant antigens (i.e., antigens not observed in mice and therefore not expected to elicit a significant response at titration) and relevant antigens present in the HLA-DQ (in-slot peptide) background were determined using ELISA. Ninety-six-well microtiter plates (Thermo Scientific) were coated overnight with a tagged pMHC complex in phosphate-buffered saline (PBS, Irwin Scientific), the complex containing relevant in-slot peptides or irrelevant antigens present in the HLA-DQ background. The plates were washed with phosphate-buffered saline containing 0.05% Tween 20 (PBS-T, Sigma-Aldrich) and blocked with bovine serum albumin (BSA, Sigma-Aldrich) in PBS.

[0224] Pre-immune and antiserum were serially diluted in BSA-PBS and added to the plate. The plate was washed, and a secondary antibody conjugated with anti-mouse IgG-Fc-horseradish peroxidase (HRP) was added. The plate was washed and developed using 3,3',5,5'-tetramethylbenzidine (TMB) / H2O2 as substrate according to the manufacturer's recommended procedure, and the absorbance at 450 nm was recorded using a spectrophotometer (Victor, PerkinElmer). Antibody titers were calculated using Graphpad PRISM software. Antibody titers were calculated as interpolated serum dilution factors with a binding signal twice that of the background.

[0225] Compared with control mice that were not tolerant to human HLA class II molecules, tolerant mice to human HLA class II molecules or a portion thereof enhanced the mice’s ability to generate specific antibody responses to pMHCs of interest. sequence list <110> Regeneron Pharmaceuticals, Inc. <120> Peptide-MHC II protein constructs and their applications <130> 057766 / 696193 <150> US 62 / 942,344 <151> 2019-12-02 <160> 73 <170> PatentIn version 3.5 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 1 Ser Gly Gly Gly Gly Gly 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 2 Gly Ser Gly Gly Ser 1 5 <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 3 Gly Gly Gly Ser 1 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 4 Gly Gly Gly Gly Ser 1 5 <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 5 Gly Gly Ser Gly 1 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 6 Gly Gly Ser Gly Gly 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 7 Gly Ser Gly Ser Gly 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 8 Gly Ser Gly Gly Gly 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 9 Gly Gly Gly Ser Gly 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 10 Gly Ser Ser Ser Gly 1 5 <210> 11 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 11 Ser Gly Gly Gly Gly Gly 1 5 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 12 Gly Cys Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 13 Gly Cys Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 14 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 15 Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 16 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 17 Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser 1 5 10 <210> 18 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 18 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 19 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 19 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 20 Gly Cys Gly Gly Ser 1 5 <210> twenty one <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> twenty one Gly Cys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> twenty two <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> twenty two Glu Asn Leu Tyr Phe Gln 1 5 <210> twenty three <211> 39 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> twenty three Leu Thr Asp Thr Leu Gln Ala Glu Thr Asp Gln Leu Glu Asp Glu Lys 1 5 10 15 Ser Ala Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu Lys Glu Lys 20 25 30 Leu Glu Phe Ile Leu Ala Ala 35 <210> twenty four <211> 40 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> twenty four Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn 1 5 10 15 Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln 20 25 30 Leu Lys Gln Lys Val Met Asn His 35 40 <210> 25 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 25 Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala 1 5 10 <210> 26 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 26 Thr Met Phe Glu Ala Leu Pro His Ile Ile Asp Glu Val Ile Asn 1 5 10 15 <210> 27 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 27 Gly Ile Lys Ala Val Tyr Asn Phe Ala Thr Cys Gly Ile Phe Ala 1 5 10 15 <210> 28 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 28 Asp Ile Tyr Lys Gly Val Tyr Gln Phe Lys Ser Val Glu Phe Asp 1 5 10 15 <210> 29 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 29 Thr Ser Ala Phe Asn Lys Lys Thr Phe Asp His Thr Leu Met Ser 1 5 10 15 <210> 30 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 30 Asp Ala Gln Ser Ala Gln Ser Gln Cys Arg Thr Phe Arg Gly Arg 1 5 10 15 <210> 31 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 31 Thr Phe Arg Gly Arg Val Leu Asp Met Phe Arg Thr Ala Phe Gly 1 5 10 15 <210> 32 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 32 Cys Asp Met Leu Arg Leu Ile Asp Tyr Asn Lys Ala Ala Leu Ser 1 5 10 15 <210> 33 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 33 Ile Glu Gln Glu Ala Asp Asn Met Ile Thr Glu Met Leu Arg Lys 1 5 10 15 <210> 34 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 34 Glu Val Lys Ser Phe Gln Trp Thr Gln Ala Leu Arg Arg Glu Leu 1 5 10 15 <210> 35 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 35 Lys Asn Val Leu Lys Val Gly Arg Leu Ser Ala Glu Glu Leu Met 1 5 10 15 <210> 36 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 36 Ser Glu Arg Pro Gln Ala Ser Gly Val Tyr Met Gly Asn Leu Thr 1 5 10 15 <210> 37 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 37 Pro Ser Leu Thr Met Ala Cys Met Ala Lys Gln Ser Gln Thr Pro 1 5 10 15 <210> 38 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 38 Glu Gly Trp Pro Tyr Ile Ala Cys Arg Thr Ser Ile Val Gly Arg 1 5 10 15 <210> 39 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 39 Ser Gln Asn Arg Lys Asp Ile Lys Leu Ile Asp Val Glu Met Thr 1 5 10 15 <210> 40 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 40 Gly Trp Leu Cys Lys Met His Thr Gly Ile Val Arg Asp Lys Lys 1 5 10 15 <210> 41 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 41 Ser Cys Lys Ser Cys Trp Gln Lys Phe Asp Ser Leu Val Arg Cys 1 5 10 15 <210> 42 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 42 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 1 5 10 15 <210> 43 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 43 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Glu Gln Lys Leu Ile Ser 1 5 10 15 Glu Glu Asp Leu His His His His His 20 25 <210> 44 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 44 Gln Leu Gln Pro Phe Pro Gln Pro Glu Leu Pro Tyr 1 5 10 <210> 45 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 45 Phe Pro Gln Pro Glu Gln Pro Phe Pro Trp Gln Pro 1 5 10 <210> 46 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 46 Pro Gln Pro Glu Leu Pro Tyr Pro Gln Pro Gln Leu 1 5 10 <210> 47 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 47 Gly Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln Gly Gly Gly Gly Ser 1 5 10 15 <210> 48 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 48 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Gly Glu Gln Lys Leu 1 5 10 15 Ile Ser Glu Glu Asp Leu His His His His His His 20 25 <210> 49 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 49 Met Ile Leu Asn Lys Ala Leu Met Leu Gly Ala Leu Ala Leu Thr Thr 1 5 10 15 Val Met Ser Pro Cys Gly Gly Glu Asp Ile Val Ala Asp His Val Ala 20 25 30 Ser Tyr Gly Val Asn Leu Tyr Gln Ser Tyr Gly Pro Ser Gly Gln Tyr 35 40 45 Thr His Glu Phe Asp Gly Asp Glu Gln Phe Tyr Val Asp Leu Gly Arg 50 55 60 Lys Glu Thr Val Trp Cys Leu Pro Val Leu Arg Gln Phe Arg Phe Asp 65 70 75 80 Pro Gln Phe Ala Leu Thr Asn Ile Ala Val Leu Lys His Asn Leu Asn 85 90 95 Ser Leu Ile Lys Arg Ser Asn Ser Thr Ala Ala Thr Asn Glu Val Pro 100 105 110 Glu Val Thr Val Phe Ser Lys Ser Pro Val Thr Leu Gly Gln Pro Asn 115 120 125 Ile Leu Ile Cys Leu Val Asp Asn Ile Phe Pro Pro Val Val Asn Ile 130 135 140 Thr Trp Leu Ser Asn Gly His Ser Val Thr Glu Gly Val Ser Glu Thr 145 150 155 160 Ser Phe Leu Ser Lys Ser Asp His Ser Phe Phe Lys Ile Ser Tyr Leu 165 170 175 Thr Leu Leu Pro Ser Ala Glu Glu Ser Tyr Asp Cys Lys Val Glu His 180 185 190 Trp Gly Leu Asp Lys Pro Leu Leu Lys His Trp Glu Pro Glu Ile Pro 195 200 205 Ala Pro Met Ser Glu Leu Thr Glu Thr Val Val Cys Ala Leu Gly Leu 210 215 220 Ser Val Gly Leu Val Gly Ile Val Val Gly Thr Val Phe Ile Ile Arg 225 230 235 240 Gly Leu Arg Ser Val Gly Ala Ser Arg His Gln Gly Pro Leu 245 250 <210> 50 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 50 Met Ser Trp Lys Lys Ala Leu Arg Ile Pro Gly Gly Leu Arg Ala Ala 1 5 10 15 Thr Val Thr Leu Met Leu Ser Met Leu Ser Thr Pro Val Ala Glu Gly 20 25 30 Arg Asp Ser Pro Glu Asp Phe Val Tyr Gln Phe Lys Gly Met Cys Tyr 35 40 45 Phe Thr Asn Gly Thr Glu Arg Val Arg Leu Val Ser Arg Ser Ile Tyr 50 55 60 Asn Arg Glu Glu Ile Val Arg Phe Asp Ser Asp Val Gly Glu Phe Arg 65 70 75 80 Ala Val Thr Leu Leu Gly Leu Pro Ala Ala Glu Tyr Trp Asn Ser Gln 85 90 95 Lys Asp Ile Leu Glu Arg Lys Arg Ala Ala Val Asp Arg Val Cys Arg 100 105 110 His Asn Tyr Gln Leu Glu Leu Arg Thr Thr Leu Gln Arg Arg Val Glu 115 120 125 Pro Thr Val Thr Ile Ser Pro Ser Arg Thr Glu Ala Leu Asn His His 130 135 140 Asn Leu Leu Val Cys Ser Val Thr Asp Phe Tyr Pro Ala Gln Ile Lys 145 150 155 160 Val Arg Trp Phe Arg Asn Asp Gln Glu Glu Thr Ala Gly Val Val Ser 165 170 175 Thr Pro Leu Ile Arg Asn Gly Asp Trp Thr Phe Gln Ile Leu Val Met 180 185 190 Leu Glu Met Thr Pro Gln Arg Gly Asp Val Tyr Thr Cys His Val Glu 195 200 205 His Pro Ser Leu Gln Ser Pro Ile Thr Val Glu Trp Arg Ala Gln Ser 210 215 220 Glu Ser Ala Gln Ser Lys Met Leu Ser Gly Ile Gly Gly Phe Val Leu 225 230 235 240 Gly Leu Ile Phe Leu Gly Leu Gly Leu Ile Ile His His Arg Ser Gln 245 250 255 Lys Gly Leu Leu His 260 <210> 51 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 51 Met Arg Pro Glu Asp Arg Met Phe His Ile Arg Ala Val Ile Leu Arg 1 5 10 15 Ala Leu Ser Leu Ala Phe Leu Leu Ser Leu Arg Gly Ala Gly Ala Ile 20 25 30 Lys Ala Asp His Val Ser Thr Tyr Ala Ala Phe Val Gln Thr His Arg 35 40 45 Pro Thr Gly Glu Phe Met Phe Glu Phe Asp Glu Asp Glu Met Phe Tyr 50 55 60 Val Asp Leu Asp Lys Lys Glu Thr Val Trp His Leu Glu Glu Phe Gly 65 70 75 80 Gln Ala Phe Ser Phe Glu Ala Gln Gly Gly Leu Ala Asn Ile Ala Ile 85 90 95 Leu Asn Asn Asn Leu Asn Thr Leu Ile Gln Arg Ser Asn His Thr Gln 100 105 110 Ala Thr Asn Asp Pro Pro Glu Val Thr Val Phe Pro Lys Glu Pro Val 115 120 125 Glu Leu Gly Gln Pro Asn Thr Leu Ile Cys His Ile Asp Lys Phe Phe 130 135 140 Pro Pro Val Leu Asn Val Thr Trp Leu Cys Asn Gly Glu Leu Val Thr 145 150 155 160 Glu Gly Val Ala Glu Ser Leu Phe Leu Pro Arg Thr Asp Tyr Ser Phe 165 170 175 His Lys Phe His Tyr Leu Thr Phe Val Pro Ser Ala Glu Asp Phe Tyr 180 185 190 Asp Cys Arg Val Glu His Trp Gly Leu Asp Gln Pro Leu Leu Lys His 195 200 205 Trp Glu Ala Gln Glu Pro Ile Gln Met Pro Glu Thr Thr Glu Thr Val 210 215 220 Leu Cys Ala Leu Gly Leu Val Leu Gly Leu Val Gly Ile Ile Val Gly 225 230 235 240 Thr Val Leu Ile Ile Lys Ser Leu Arg Ser Gly His Asp Pro Arg Ala 245 250 255 Gln Gly Thr Leu 260 <210> 52 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 52 Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val 1 5 10 15 Leu Met Ser Ala Gln Glu Ser Trp Ala Ile Lys Glu Glu His Val Ile 20 25 30 Ile Gln Ala Glu Phe Tyr Leu Asn Pro Asp Gln Ser Gly Glu Phe Met 35 40 45 Phe Asp Phe Asp Gly Asp Glu Ile Phe His Val Asp Met Ala Lys Lys 50 55 60 Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu 65 70 75 80 Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala Asn Leu Glu 85 90 95 Ile Met Thr Lys Arg Ser Asn Tyr Thr Pro Ile Thr Asn Val Pro Pro 100 105 110 Glu Val Thr Val Leu Thr Asn Ser Pro Val Glu Leu Arg Glu Pro Asn 115 120 125 Val Leu Ile Cys Phe Ile Asp Lys Phe Thr Pro Pro Val Val Asn Val 130 135 140 Thr Trp Leu Arg Asn Gly Lys Pro Val Thr Thr Gly Val Ser Glu Thr 145 150 155 160 Val Phe Leu Pro Arg Glu Asp His Leu Phe Arg Lys Phe His Tyr Leu 165 170 175 Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr Asp Cys Arg Val Glu His 180 185 190 Trp Gly Leu Asp Glu Pro Leu Leu Lys His Trp Glu Phe Asp Ala Pro 195 200 205 Ser Pro Leu Pro Glu Thr Thr Glu Asn Val Val Cys Ala Leu Gly Leu 210 215 220 Thr Val Gly Leu Val Gly Ile Ile Ile Gly Thr Ile Phe Ile Ile Lys 225 230 235 240 Gly Val Arg Lys Ser Asn Ala Ala Glu Arg Arg Gly Pro Leu 245 250 <210> 53 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 53 Met Ile Leu Asn Lys Ala Leu Met Leu Gly Ala Leu Ala Leu Thr Thr 1 5 10 15 Val Met Ser Pro Cys Gly Gly Glu Asp Ile Val Ala Asp His Val Ala 20 25 30 Ser Tyr Gly Val Asn Leu Tyr Gln Ser Tyr Gly Pro Ser Gly Gln Tyr 35 40 45 Thr His Glu Phe Asp Gly Asp Glu Gln Phe Tyr Val Asp Leu Gly Arg 50 55 60 Lys Glu Thr Val Trp Cys Leu Pro Val Leu Arg Gln Phe Arg Phe Asp 65 70 75 80 Pro Gln Phe Ala Leu Thr Asn Ile Ala Val Leu Lys His Asn Leu Asn 85 90 95 Ser Leu Ile Lys Cys Ser Asn Ser Thr Ala Ala Thr Asn Glu Val Pro 100 105 110 Glu Val Thr Val Phe Ser Lys Ser Pro Val Thr Leu Gly Gln Pro Asn 115 120 125 Ile Leu Ile Cys Leu Val Asp Asn Ile Phe Pro Pro Val Val Asn Ile 130 135 140 Thr Trp Leu Ser Asn Gly His Ser Val Thr Glu Gly Val Ser Glu Thr 145 150 155 160 Ser Phe Leu Ser Lys Ser Asp His Ser Phe Phe Lys Ile Ser Tyr Leu 165 170 175 Thr Leu Leu Pro Ser Ala Glu Glu Ser Tyr Asp Cys Lys Val Glu His 180 185 190 Trp Gly Leu Asp Lys Pro Leu Leu Lys His Trp Glu Pro Glu Ile Pro 195 200 205 Ala Pro Met Ser Glu Leu Thr Glu Thr Val Val Cys Ala Leu Gly Leu 210 215 220 Ser Val Gly Leu Val Gly Ile Val Val Gly Thr Val Phe Ile Ile Arg 225 230 235 240 Gly Leu Arg Ser Val Gly Ala Ser Arg His Gln Gly Pro Leu 245 250 <210> 54 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 54 Met Ile Leu Asn Lys Ala Leu Met Leu Gly Ala Leu Ala Leu Thr Thr 1 5 10 15 Val Met Ser Pro Cys Gly Gly Glu Asp Ile Val Ala Asp His Val Ala 20 25 30 Ser Tyr Gly Val Asn Leu Tyr Gln Ser Tyr Gly Pro Ser Gly Gln Tyr 35 40 45 Thr His Glu Phe Asp Gly Asp Glu Gln Phe Tyr Val Asp Leu Gly Arg 50 55 60 Lys Glu Thr Val Trp Ala Leu Pro Val Leu Arg Gln Phe Arg Phe Asp 65 70 75 80 Pro Gln Phe Ala Leu Thr Asn Ile Ala Val Leu Lys His Asn Leu Asn 85 90 95 Ser Leu Ile Lys Cys Ser Asn Ser Thr Ala Ala Thr Asn Glu Val Pro 100 105 110 Glu Val Thr Val Phe Ser Lys Ser Pro Val Thr Leu Gly Gln Pro Asn 115 120 125 Ile Leu Ile Cys Leu Val Asp Asn Ile Phe Pro Pro Val Val Asn Ile 130 135 140 Thr Trp Leu Ser Asn Gly His Ser Val Thr Glu Gly Val Ser Glu Thr 145 150 155 160 Ser Phe Leu Ser Lys Ser Asp His Ser Phe Phe Lys Ile Ser Tyr Leu 165 170 175 Thr Leu Leu Pro Ser Ala Glu Glu Ser Tyr Asp Cys Lys Val Glu His 180 185 190 Trp Gly Leu Asp Lys Pro Leu Leu Lys His Trp Glu Pro Glu Ile Pro 195 200 205 Ala Pro Met Ser Glu Leu Thr Glu Thr Val Val Cys Ala Leu Gly Leu 210 215 220 Ser Val Gly Leu Val Gly Ile Val Val Gly Thr Val Phe Ile Ile Arg 225 230 235 240 Gly Leu Arg Ser Val Gly Ala Ser Arg His Gln Gly Pro Leu 245 250 <210> 55 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 55 Met Ile Leu Asn Lys Ala Leu Met Leu Gly Ala Leu Ala Leu Thr Thr 1 5 10 15 Val Met Ser Pro Cys Gly Gly Glu Asp Ile Val Ala Asp His Val Ala 20 25 30 Ser Tyr Gly Val Asn Leu Tyr Gln Ser Tyr Gly Pro Ser Gly Gln Tyr 35 40 45 Thr His Glu Phe Asp Gly Asp Glu Gln Phe Tyr Val Asp Leu Gly Arg 50 55 60 Lys Glu Thr Val Trp Gln Leu Pro Val Leu Arg Gln Phe Arg Phe Asp 65 70 75 80 Pro Gln Phe Ala Leu Thr Asn Ile Ala Val Leu Lys His Asn Leu Asn 85 90 95 Ser Leu Ile Lys Arg Ser Asn Ser Thr Ala Ala Thr Asn Glu Val Pro 100 105 110 Glu Val Thr Val Phe Ser Lys Ser Pro Val Thr Leu Gly Gln Pro Asn 115 120 125 Ile Leu Ile Cys Leu Val Asp Asn Ile Phe Pro Pro Val Val Asn Ile 130 135 140 Thr Trp Leu Ser Asn Gly His Ser Val Thr Glu Gly Val Ser Glu Thr 145 150 155 160 Ser Phe Leu Ser Lys Ser Asp His Ser Phe Phe Lys Ile Ser Tyr Leu 165 170 175 Thr Leu Leu Pro Ser Ala Glu Glu Ser Tyr Asp Cys Lys Val Glu His 180 185 190 Trp Gly Leu Asp Lys Pro Leu Leu Lys His Trp Glu Pro Glu Ile Pro 195 200 205 Ala Pro Met Ser Glu Leu Thr Glu Thr Val Val Cys Ala Leu Gly Leu 210 215 220 Ser Val Gly Leu Val Gly Ile Val Val Gly Thr Val Phe Ile Ile Arg 225 230 235 240 Gly Leu Arg Ser Val Gly Ala Ser Arg His Gln Gly Pro Leu 245 250 <210> 56 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 56 Met Ile Leu Asn Lys Ala Leu Met Leu Gly Ala Leu Ala Leu Thr Thr 1 5 10 15 Val Met Ser Pro Cys Gly Gly Glu Asp Ile Val Ala Asp His Val Ala 20 25 30 Ser Tyr Gly Val Asn Leu Tyr Gln Ser Tyr Gly Pro Ser Gly Gln Tyr 35 40 45 Thr His Glu Phe Asp Gly Asp Glu Gln Phe Tyr Val Asp Leu Gly Arg 50 55 60 Lys Glu Thr Val Trp Ala Leu Pro Val Leu Arg Gln Phe Arg Phe Asp 65 70 75 80 Pro Gln Phe Ala Leu Thr Asn Ile Ala Val Leu Lys His Asn Leu Asn 85 90 95 Ser Leu Ile Lys Arg Ser Asn Ser Thr Ala Ala Thr Asn Glu Val Pro 100 105 110 Glu Val Thr Val Phe Ser Lys Ser Pro Val Thr Leu Gly Gln Pro Asn 115 120 125 Ile Leu Ile Cys Leu Val Asp Asn Ile Phe Pro Pro Val Val Asn Ile 130 135 140 Thr Trp Leu Ser Asn Gly His Ser Val Thr Glu Gly Val Ser Glu Thr 145 150 155 160 Ser Phe Leu Ser Lys Ser Asp His Ser Phe Phe Lys Ile Ser Tyr Leu 165 170 175 Thr Leu Leu Pro Ser Ala Glu Glu Ser Tyr Asp Cys Lys Val Glu His 180 185 190 Trp Gly Leu Asp Lys Pro Leu Leu Lys His Trp Glu Pro Glu Ile Pro 195 200 205 Ala Pro Met Ser Glu Leu Thr Glu Thr Val Val Cys Ala Leu Gly Leu 210 215 220 Ser Val Gly Leu Val Gly Ile Val Val Gly Thr Val Phe Ile Ile Arg 225 230 235 240 Gly Leu Arg Ser Val Gly Ala Ser Arg His Gln Gly Pro Leu 245 250 <210> 57 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 57 Met Ile Leu Asn Lys Ala Leu Met Leu Gly Ala Leu Ala Leu Thr Thr 1 5 10 15 Val Met Ser Pro Cys Gly Gly Glu Asp Ile Val Ala Asp His Val Ala 20 25 30 Ser Tyr Gly Val Asn Leu Tyr Gln Ser Tyr Gly Pro Ser Gly Gln Tyr 35 40 45 Thr His Glu Phe Asp Gly Asp Glu Gln Phe Tyr Val Asp Leu Gly Arg 50 55 60 Lys Glu Thr Val Trp Gln Leu Pro Val Leu Arg Gln Phe Arg Phe Asp 65 70 75 80 Pro Gln Phe Ala Leu Thr Asn Ile Ala Val Leu Lys His Asn Leu Asn 85 90 95 Ser Leu Ile Lys Cys Ser Asn Ser Thr Ala Ala Thr Asn Glu Val Pro 100 105 110 Glu Val Thr Val Phe Ser Lys Ser Pro Val Thr Leu Gly Gln Pro Asn 115 120 125 Ile Leu Ile Cys Leu Val Asp Asn Ile Phe Pro Pro Val Val Asn Ile 130 135 140 Thr Trp Leu Ser Asn Gly His Ser Val Thr Glu Gly Val Ser Glu Thr 145 150 155 160 Ser Phe Leu Ser Lys Ser Asp His Ser Phe Phe Lys Ile Ser Tyr Leu 165 170 175 Thr Leu Leu Pro Ser Ala Glu Glu Ser Tyr Asp Cys Lys Val Glu His 180 185 190 Trp Gly Leu Asp Lys Pro Leu Leu Lys His Trp Glu Pro Glu Ile Pro 195 200 205 Ala Pro Met Ser Glu Leu Thr Glu Thr Val Val Cys Ala Leu Gly Leu 210 215 220 Ser Val Gly Leu Val Gly Ile Val Val Gly Thr Val Phe Ile Ile Arg 225 230 235 240 Gly Leu Arg Ser Val Gly Ala Ser Arg His Gln Gly Pro Leu 245 250 <210> 58 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 58 Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val 1 5 10 15 Leu Met Ser Ala Gln Glu Ser Trp Ala Ile Lys Glu Glu His Val Ile 20 25 30 Ile Gln Ala Glu Phe Tyr Leu Asn Pro Asp Gln Ser Gly Glu Phe Met 35 40 45 Phe Asp Phe Asp Gly Asp Glu Ile Phe His Val Asp Met Ala Lys Lys 50 55 60 Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Cys Glu 65 70 75 80 Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys Ala Asn Leu Glu 85 90 95 Ile Met Thr Lys Arg Ser Asn Tyr Thr Pro Ile Thr Asn Val Pro Pro 100 105 110 Glu Val Thr Val Leu Thr Asn Ser Pro Val Glu Leu Arg Glu Pro Asn 115 120 125 Val Leu Ile Cys Phe Ile Asp Lys Phe Thr Pro Pro Val Val Asn Val 130 135 140 Thr Trp Leu Arg Asn Gly Lys Pro Val Thr Thr Gly Val Ser Glu Thr 145 150 155 160 Val Phe Leu Pro Arg Glu Asp His Leu Phe Arg Lys Phe His Tyr Leu 165 170 175 Pro Phe Leu Pro Ser Thr Glu Asp Val Tyr Asp Cys Arg Val Glu His 180 185 190 Trp Gly Leu Asp Glu Pro Leu Leu Lys His Trp Glu Phe Asp Ala Pro 195 200 205 Ser Pro Leu Pro Glu Thr Thr Glu Asn Val Val Cys Ala Leu Gly Leu 210 215 220 Thr Val Gly Leu Val Gly Ile Ile Ile Gly Thr Ile Phe Ile Ile Lys 225 230 235 240 Gly Val Arg Lys Ser Asn Ala Ala Glu Arg Arg Gly Pro Leu 245 250 <210> 59 <211> 193 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 59 Glu Asp Ile Val Ala Asp His Val Ala Ser Tyr Gly Val Asn Leu Tyr 1 5 10 15 Gln Ser Tyr Gly Pro Ser Gly Gln Tyr Thr His Glu Phe Asp Gly Asp 20 25 30 Glu Gln Phe Tyr Val Asp Leu Gly Arg Lys Glu Thr Val Trp Cys Leu 35 40 45 Pro Val Leu Arg Gln Phe Arg Phe Asp Pro Gln Phe Ala Leu Thr Asn 50 55 60 Ile Ala Val Leu Lys His Asn Leu Asn Ser Leu Ile Lys Arg Ser Asn 65 70 75 80 Ser Thr Ala Ala Thr Asn Glu Val Pro Glu Val Thr Val Phe Ser Lys 85 90 95 Ser Pro Val Thr Leu Gly Gln Pro Asn Ile Leu Ile Cys Leu Val Asp 100 105 110 Asn Ile Phe Pro Pro Val Val Asn Ile Thr Trp Leu Ser Asn Gly His 115 120 125 Ser Val Thr Glu Gly Val Ser Glu Thr Ser Phe Leu Ser Lys Ser Asp 130 135 140 His Ser Phe Phe Lys Ile Ser Tyr Leu Thr Leu Leu Pro Ser Ala Glu 145 150 155 160 Glu Ser Tyr Asp Cys Lys Val Glu His Trp Gly Leu Asp Lys Pro Leu 165 170 175 Leu Lys His Trp Glu Pro Glu Ile Pro Ala Pro Met Ser Glu Leu Thr 180 185 190 Glu <210> 60 <211> 198 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 60 Arg Asp Ser Pro Glu Asp Phe Val Tyr Gln Phe Lys Gly Met Cys Tyr 1 5 10 15 Phe Thr Asn Gly Thr Glu Arg Val Arg Leu Val Ser Arg Ser Ile Tyr 20 25 30 Asn Arg Glu Glu Ile Val Arg Phe Asp Ser Asp Val Gly Glu Phe Arg 35 40 45 Ala Val Thr Leu Leu Gly Leu Pro Ala Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Ile Leu Glu Arg Lys Arg Ala Ala Val Asp Arg Val Cys Arg 65 70 75 80 His Asn Tyr Gln Leu Glu Leu Arg Thr Thr Leu Gln Arg Arg Val Glu 85 90 95 Pro Thr Val Thr Ile Ser Pro Ser Arg Thr Glu Ala Leu Asn His His 100 105 110 Asn Leu Leu Val Cys Ser Val Thr Asp Phe Tyr Pro Ala Gln Ile Lys 115 120 125 Val Arg Trp Phe Arg Asn Asp Gln Glu Glu Thr Ala Gly Val Val Ser 130 135 140 Thr Pro Leu Ile Arg Asn Gly Asp Trp Thr Phe Gln Ile Leu Val Met 145 150 155 160 Leu Glu Met Thr Pro Gln Arg Gly Asp Val Tyr Thr Cys His Val Glu 165 170 175 His Pro Ser Leu Gln Ser Pro Ile Thr Val Glu Trp Arg Ala Gln Ser 180 185 190 Glu Ser Ala Gln Ser Lys 195 <210> 61 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 61 Ala Gly Ala Ile Lys Ala Asp His Val Ser Thr Tyr Ala Ala Phe Val 1 5 10 15 Gln Thr His Arg Pro Thr Gly Glu Phe Met Phe Glu Phe Asp Glu Asp 20 25 30 Glu Met Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr Val Trp His Leu 35 40 45 Glu Glu Phe Gly Gln Ala Phe Ser Phe Glu Ala Gln Gly Gly Leu Ala 50 55 60 Asn Ile Ala Ile Leu Asn Asn Asn Leu Asn Thr Leu Ile Gln Arg Ser 65 70 75 80 Asn His Thr Gln Ala Thr Asn Asp Pro Pro Glu Val Thr Val Phe Pro 85 90 95 Lys Glu Pro Val Glu Leu Gly Gln Pro Asn Thr Leu Ile Cys His Ile 100 105 110 Asp Lys Phe Phe Pro Pro Val Leu Asn Val Thr Trp Leu Cys Asn Gly 115 120 125 Glu Leu Val Thr Glu Gly Val Ala Glu Ser Leu Phe Leu Pro Arg Thr 130 135 140 Asp Tyr Ser Phe His Lys Phe His Tyr Leu Thr Phe Val Pro Ser Ala 145 150 155 160 Glu Asp Phe Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Gln Pro 165 170 175 Leu Leu Lys His Trp Glu Ala Gln Glu Pro Ile Gln Met Pro Glu Thr 180 185 190 Thr Glu <210> 62 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 62 Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro 1 5 10 15 Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe 20 25 30 His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe 35 40 45 Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala 50 55 60 Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr 65 70 75 80 Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro 85 90 95 Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe 100 105 110 Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val 115 120 125 Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu 130 135 140 Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val 145 150 155 160 Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys 165 170 175 His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu 180 185 190 <210> 63 <211> 193 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 63 Glu Asp Ile Val Ala Asp His Val Ala Ser Tyr Gly Val Asn Leu Tyr 1 5 10 15 Gln Ser Tyr Gly Pro Ser Gly Gln Tyr Thr His Glu Phe Asp Gly Asp 20 25 30 Glu Gln Phe Tyr Val Asp Leu Gly Arg Lys Glu Thr Val Trp Cys Leu 35 40 45 Pro Val Leu Arg Gln Phe Arg Phe Asp Pro Gln Phe Ala Leu Thr Asn 50 55 60 Ile Ala Val Leu Lys His Asn Leu Asn Ser Leu Ile Lys Cys Ser Asn 65 70 75 80 Ser Thr Ala Ala Thr Asn Glu Val Pro Glu Val Thr Val Phe Ser Lys 85 90 95 Ser Pro Val Thr Leu Gly Gln Pro Asn Ile Leu Ile Cys Leu Val Asp 100 105 110 Asn Ile Phe Pro Pro Val Val Asn Ile Thr Trp Leu Ser Asn Gly His 115 120 125 Ser Val Thr Glu Gly Val Ser Glu Thr Ser Phe Leu Ser Lys Ser Asp 130 135 140 His Ser Phe Phe Lys Ile Ser Tyr Leu Thr Leu Leu Pro Ser Ala Glu 145 150 155 160 Glu Ser Tyr Asp Cys Lys Val Glu His Trp Gly Leu Asp Lys Pro Leu 165 170 175 Leu Lys His Trp Glu Pro Glu Ile Pro Ala Pro Met Ser Glu Leu Thr 180 185 190 Glu <210> 64 <211> 193 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 64 Glu Asp Ile Val Ala Asp His Val Ala Ser Tyr Gly Val Asn Leu Tyr 1 5 10 15 Gln Ser Tyr Gly Pro Ser Gly Gln Tyr Thr His Glu Phe Asp Gly Asp 20 25 30 Glu Gln Phe Tyr Val Asp Leu Gly Arg Lys Glu Thr Val Trp Ala Leu 35 40 45 Pro Val Leu Arg Gln Phe Arg Phe Asp Pro Gln Phe Ala Leu Thr Asn 50 55 60 Ile Ala Val Leu Lys His Asn Leu Asn Ser Leu Ile Lys Cys Ser Asn 65 70 75 80 Ser Thr Ala Ala Thr Asn Glu Val Pro Glu Val Thr Val Phe Ser Lys 85 90 95 Ser Pro Val Thr Leu Gly Gln Pro Asn Ile Leu Ile Cys Leu Val Asp 100 105 110 Asn Ile Phe Pro Pro Val Val Asn Ile Thr Trp Leu Ser Asn Gly His 115 120 125 Ser Val Thr Glu Gly Val Ser Glu Thr Ser Phe Leu Ser Lys Ser Asp 130 135 140 His Ser Phe Phe Lys Ile Ser Tyr Leu Thr Leu Leu Pro Ser Ala Glu 145 150 155 160 Glu Ser Tyr Asp Cys Lys Val Glu His Trp Gly Leu Asp Lys Pro Leu 165 170 175 Leu Lys His Trp Glu Pro Glu Ile Pro Ala Pro Met Ser Glu Leu Thr 180 185 190 Glu <210> 65 <211> 193 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 65 Glu Asp Ile Val Ala Asp His Val Ala Ser Tyr Gly Val Asn Leu Tyr 1 5 10 15 Gln Ser Tyr Gly Pro Ser Gly Gln Tyr Thr His Glu Phe Asp Gly Asp 20 25 30 Glu Gln Phe Tyr Val Asp Leu Gly Arg Lys Glu Thr Val Trp Gln Leu 35 40 45 Pro Val Leu Arg Gln Phe Arg Phe Asp Pro Gln Phe Ala Leu Thr Asn 50 55 60 Ile Ala Val Leu Lys His Asn Leu Asn Ser Leu Ile Lys Arg Ser Asn 65 70 75 80 Ser Thr Ala Ala Thr Asn Glu Val Pro Glu Val Thr Val Phe Ser Lys 85 90 95 Ser Pro Val Thr Leu Gly Gln Pro Asn Ile Leu Ile Cys Leu Val Asp 100 105 110 Asn Ile Phe Pro Pro Val Val Asn Ile Thr Trp Leu Ser Asn Gly His 115 120 125 Ser Val Thr Glu Gly Val Ser Glu Thr Ser Phe Leu Ser Lys Ser Asp 130 135 140 His Ser Phe Phe Lys Ile Ser Tyr Leu Thr Leu Leu Pro Ser Ala Glu 145 150 155 160 Glu Ser Tyr Asp Cys Lys Val Glu His Trp Gly Leu Asp Lys Pro Leu 165 170 175 Leu Lys His Trp Glu Pro Glu Ile Pro Ala Pro Met Ser Glu Leu Thr 180 185 190 Glu <210> 66 <211> 193 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 66 Glu Asp Ile Val Ala Asp His Val Ala Ser Tyr Gly Val Asn Leu Tyr 1 5 10 15 Gln Ser Tyr Gly Pro Ser Gly Gln Tyr Thr His Glu Phe Asp Gly Asp 20 25 30 Glu Gln Phe Tyr Val Asp Leu Gly Arg Lys Glu Thr Val Trp Ala Leu 35 40 45 Pro Val Leu Arg Gln Phe Arg Phe Asp Pro Gln Phe Ala Leu Thr Asn 50 55 60 Ile Ala Val Leu Lys His Asn Leu Asn Ser Leu Ile Lys Arg Ser Asn 65 70 75 80 Ser Thr Ala Ala Thr Asn Glu Val Pro Glu Val Thr Val Phe Ser Lys 85 90 95 Ser Pro Val Thr Leu Gly Gln Pro Asn Ile Leu Ile Cys Leu Val Asp 100 105 110 Asn Ile Phe Pro Pro Val Val Asn Ile Thr Trp Leu Ser Asn Gly His 115 120 125 Ser Val Thr Glu Gly Val Ser Glu Thr Ser Phe Leu Ser Lys Ser Asp 130 135 140 His Ser Phe Phe Lys Ile Ser Tyr Leu Thr Leu Leu Pro Ser Ala Glu 145 150 155 160 Glu Ser Tyr Asp Cys Lys Val Glu His Trp Gly Leu Asp Lys Pro Leu 165 170 175 Leu Lys His Trp Glu Pro Glu Ile Pro Ala Pro Met Ser Glu Leu Thr 180 185 190 Glu <210> 67 <211> 193 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 67 Glu Asp Ile Val Ala Asp His Val Ala Ser Tyr Gly Val Asn Leu Tyr 1 5 10 15 Gln Ser Tyr Gly Pro Ser Gly Gln Tyr Thr His Glu Phe Asp Gly Asp 20 25 30 Glu Gln Phe Tyr Val Asp Leu Gly Arg Lys Glu Thr Val Trp Gln Leu 35 40 45 Pro Val Leu Arg Gln Phe Arg Phe Asp Pro Gln Phe Ala Leu Thr Asn 50 55 60 Ile Ala Val Leu Lys His Asn Leu Asn Ser Leu Ile Lys Cys Ser Asn 65 70 75 80 Ser Thr Ala Ala Thr Asn Glu Val Pro Glu Val Thr Val Phe Ser Lys 85 90 95 Ser Pro Val Thr Leu Gly Gln Pro Asn Ile Leu Ile Cys Leu Val Asp 100 105 110 Asn Ile Phe Pro Pro Val Val Asn Ile Thr Trp Leu Ser Asn Gly His 115 120 125 Ser Val Thr Glu Gly Val Ser Glu Thr Ser Phe Leu Ser Lys Ser Asp 130 135 140 His Ser Phe Phe Lys Ile Ser Tyr Leu Thr Leu Leu Pro Ser Ala Glu 145 150 155 160 Glu Ser Tyr Asp Cys Lys Val Glu His Trp Gly Leu Asp Lys Pro Leu 165 170 175 Leu Lys His Trp Glu Pro Glu Ile Pro Ala Pro Met Ser Glu Leu Thr 180 185 190 Glu <210> 68 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 68 Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro 1 5 10 15 Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe 20 25 30 His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe 35 40 45 Gly Arg Phe Ala Ser Cys Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala 50 55 60 Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr 65 70 75 80 Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro 85 90 95 Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys Phe 100 105 110 Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val 115 120 125 Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu 130 135 140 Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val 145 150 155 160 Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys 165 170 175 His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu 180 185 190 <210> 69 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 69 Gln Pro Phe Pro Gln Pro Glu Leu Pro Tyr Pro Gln 1 5 10 <210> 70 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 70 Gln Pro Phe Pro Gln Pro Glu Gln Pro Phe Pro Trp 1 5 10 <210> 71 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 71 Gln Pro Phe Pro Gln Pro Glu Leu Pro Tyr 1 5 10 <210> 72 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 72 Phe Pro Gln Pro Glu Leu Pro Tyr Pro Gln 1 5 10 <210> 73 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 73 Phe Pro Gln Pro Glu Gln Pro Phe Pro Trp 1 5 10

Claims

1. A composition comprising an MHC ligand peptide covalently attached to an MHC class II molecule, said MHC class II molecule being composed of an MHC class II α chain and an MHC class II β chain. The MHC class II molecule mentioned above is the human HLA-DQ2 molecule. The MHC ligand peptide is 10 to 12 amino acids in length, and contains gliadin αI, αII, or ω2 epitopes. The MHC ligand peptide can bind to the MHC class II molecule, and is covalently attached to the MHC class II molecule via a peptide linker. The peptide linker is a flexible linker with a length between 10 and 20 amino acids, extending from the C-terminus of the MHC ligand peptide to the N-terminus of the MHC class II β chain. The peptide linker comprises a first cysteine ​​residue, which is the only cysteine ​​residue in the peptide linker. The peptide linker comprises 2-4 repeats of the sequence listed in SEQ ID NO: 4, wherein the amino acid in the first repeat is mutated to the first cysteine ​​residue, and wherein the first cysteine ​​residue is located in the first four amino acids of the peptide linker. The MHC class II α chain contains a second cysteine ​​residue, wherein, when the MHC class II α chain is optimally aligned with SEQ ID NO: 49, the second cysteine ​​residue is located at position 101 in the sequence listed in SEQ ID NO: 49, and the MHC class II α chain lacks the cysteine ​​residue present in the corresponding wild-type MHC class II α chain, wherein the cysteine ​​residue in the corresponding wild-type MHC class II α chain is located at position 70 in the sequence listed in SEQ ID NO:

49. The first cysteine ​​and the second cysteine ​​form a disulfide bond, causing the MHC ligand peptide to bind in the peptide-binding groove formed by the MHC class II α chain and the MHC class II β chain. The MHC class II α chain is composed of the sequence listed in SEQ ID NO: 64, and the MHC class II β chain is composed of the sequence listed in SEQ ID NO:

60. The MHC class II α chain and the MHC class II β chain are connected via Jun Fos zippers, knobs Pores, or connections to the Fc region of immunoglobulins.

2. The composition according to claim 1, wherein the MHC class II α chain and the MHC class II β chain are connected by a Jun-Fos zipper comprising a Jun leucine zipper dimerization motif and a Fos leucine zipper dimerization motif, and wherein... The MHC class II α chain is connected to the Jun leucine zipper dimerization motif, and the MHC class II β chain is connected to the Fos leucine zipper dimerization motif; or the MHC class II α chain is connected to the Fos leucine zipper dimerization motif, and the MHC class II β chain is connected to the Jun leucine zipper dimerization motif.

3. The composition according to claim 2, wherein the C-terminus of the MHC class II α chain is linked to the Jun leucine zipper dimerization motif, and the C-terminus of the MHC class II β chain is linked to the Fos leucine zipper dimerization motif, or The C-terminus of the MHC class II α chain is connected to the Fos leucine zipper dimerization motif, and the C-terminus of the MHC class II β chain is connected to the Jun leucine zipper dimerization motif.

4. The composition according to claim 2, wherein the MHC class II α chain is linked to the Jun leucine zipper dimerization motif via an MHC-Jun linker, and the MHC class II β chain is linked to the Fos leucine zipper dimerization motif via an MHC-Fos linker, or The MHC class II α chain is connected to the Fos leucine zipper dimerization motif via the MHC-Fos linker, and the MHC class II β chain is connected to the Jun leucine zipper dimerization motif via the MHC-Jun linker.

5. The composition of claim 4, wherein the MHC-Jun connector and the MHC-Fos connector each comprise the sequence listed in SEQ ID NO:

1.

6. The composition according to any one of claims 1-5, wherein the MHC ligand peptide comprises the sequence shown in any one of SEQ ID NO: 44-46 and 69-73.

7. The composition according to claim 6, wherein the MHC ligand peptide comprises the sequence shown in any one of SEQ ID NO: 44-46 and 69-73.

8. The composition according to any one of claims 1-5 and 7, wherein the peptide linker connecting the MHC ligand peptide to the MHC class II molecule is 15 to 20 amino acids in length and the peptide linker comprises the sequence listed in SEQ ID NO:

21.

9. The composition of claim 8, wherein the peptide linker connecting the MHC ligand peptide to the MHC class II molecule consists of the sequence listed in SEQ ID NO:

21.

10. The composition according to any one of claims 1-5, wherein the MHC ligand peptide comprises the sequence shown in any one of SEQ ID NO:44-46 and 69-73, and The peptide linker connecting the MHC ligand peptide to the MHC class II molecule is 15 to 20 amino acids in length and contains the sequence listed in SEQ ID NO:

21.

11. The composition according to any one of claims 1-5, wherein the MHC ligand peptide comprises the sequence shown in any one of SEQ ID NO:44-46 and 69-73, and The peptide linker that connects the MHC ligand peptide to the MHC class II molecule consists of the sequence listed in SEQ ID NO:

21.

12. The composition according to any one of claims 1-5, wherein the MHC ligand peptide comprises the sequence shown in any one of SEQ ID NO: 44-46 and 69-73, and The peptide linker connecting the MHC ligand peptide to the MHC class II molecule is 15 to 20 amino acids in length and contains the sequence listed in SEQ ID NO:

21.

13. The composition according to any one of claims 1-5, wherein the MHC ligand peptide comprises the sequence shown in any one of SEQ ID NO: 44-46 and 69-73, and The peptide linker that connects the MHC ligand peptide to the MHC class II molecule consists of the sequence listed in SEQ ID NO:

21.

14. The composition according to any one of claims 1-5, 7 and 9, wherein the composition further comprises one or more immunostimulatory molecules.

15. The composition of claim 14, wherein the one or more immunostimulatory molecules comprise a pan-DR binding epitope (PADRE) and / or a peptide derived from lymphocytic choriomeningitis virus (LCMV).

16. The composition of claim 14, wherein the one or more immunostimulatory molecules are directly or indirectly covalently linked to the MHC class II molecule.

17. A nucleic acid encoding a composition according to any one of claims 1-16.

18. Use of the composition of any one of claims 1-16 or the nucleic acid encoding the composition in the preparation of an immunotherapeutic medicament, wherein the indication for the immunotherapeutic medicament is celiac disease.

19. The use according to claim 18, wherein the immunotherapeutic agent is an antigen-binding protein.

20. The use according to claim 19, wherein the antigen-binding protein specifically binds to the composition.

Images