Peptide-MHC ii protein construct and use of the same

Abstract

To provide a peptide-MHC II protein construct and use of the construct.SOLUTION: Provided is a composition includes an MHC ligand peptide covalently bonded to an MHC class II molecule. In several compositions, the MHC ligand peptide is covalently bonded to the MHC class II molecule by a peptide linker, the MHC ligand peptide or peptide linker includes first cysteine, an MHC class IIα chain or a part thereof or an MHC class IIβ chain or a part thereof includes second cysteine, the first cysteine and the second cysteine form disulfide bond such that the MHC ligand peptide bonds in a peptide-binding groove formed of the MHC class IIα chain or a part thereof and the MHC class IIβ chain or a part thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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.

[0002] Reference to sequence listings submitted as text files via EFS Web The sequence listing set forth in file 696193SEQLIST.txt is 50.9 kilobytes, was created on November 11, 2020, and is incorporated herein by reference. [Background technology]

[0003] Soluble peptide-MHC I protein constructs have been previously described. These constructs can be used for a variety of purposes, such as immunizing rodents (e.g., VELOCIMMUNE® rodents) to generate anti-peptide in-groove antibodies. However, there is a need for more soluble peptide-MHC II protein constructs. Summary of the Invention [Means for solving the problem]

[0004] Compositions comprising an MHC ligand peptide covalently linked to an MHC class II molecule, nucleic acids encoding such compositions, and methods of using such compositions to elicit an immune response in a subject are provided.

[0005] In one embodiment, a composition is provided comprising an MHC ligand peptide covalently linked to an MHC class II molecule comprising an MHC class II α chain or portion thereof and an MHC class II β chain or portion thereof. In some such compositions, the MHC ligand peptide is covalently linked to the MHC class II molecule by a peptide linker. In some such compositions, the MHC ligand peptide or peptide linker comprises a first cysteine, and the MHC class II molecule comprises a second cysteine. In some such compositions, the first and second cysteines form a disulfide bond such that the MHC ligand peptide binds to the peptide-binding groove formed by the MHC class II α chain or portion thereof and the MHC class II β chain or portion thereof.

[0006] In some such compositions, the MHC class II α chain or portion thereof comprises an α1 domain, and the MHC class II β chain or portion thereof comprises a β1 domain. Optionally, the MHC class II α chain or portion thereof comprises an MHC class II α chain extracellular domain, and the MHC class II β chain or portion thereof comprises an MHC class II β chain extracellular domain. Optionally, the MHC class II α chain or portion thereof comprises an α1 domain, an α2 domain, a transmembrane domain, and a cytoplasmic domain. Optionally, the MHC class II β chain or portion thereof comprises a β1 domain, a β2 domain, a transmembrane domain, and a cytoplasmic domain.

[0007] 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 portion thereof comprises an α1 domain and an α2 domain, but does not comprise a transmembrane domain or a cytoplasmic domain. Optionally, the MHC class II β chain or portion thereof comprises a β1 domain and a β2 domain, but does not comprise a transmembrane domain or a cytoplasmic domain. Optionally, the MHC class II α chain or portion thereof and the MHC class II β chain or portion thereof are linked by a Jun-Fos zipper, electrostatic engineering, knobs-into-holes, an immunoglobulin scaffold, an immunoglobulin Fc region, or a linker. Optionally, the MHC class II α chain or portion thereof and the MHC class II β chain or portion thereof are linked by a Jun-Fos zipper comprising a Jun leucine zipper dimerization motif and a Fos leucine zipper dimerization motif, wherein the MHC class II α chain or portion thereof is linked to the Jun leucine zipper dimerization motif and the MHC class II β chain or portion thereof is linked to the Fos leucine zipper dimerization motif, or the MHC class II α chain or portion thereof is linked to the Fos leucine zipper dimerization motif and the MHC class II β chain or portion thereof is linked to the Jun leucine zipper dimerization motif. Optionally, the C-terminus of the MHC class II α chain or portion thereof is linked to the Jun leucine zipper dimerization motif and the C-terminus of the MHC class II β chain or portion thereof is linked to the Fos leucine zipper dimerization motif. Optionally, the C-terminus of the MHC class II α chain or portion thereof is linked to a Fos leucine zipper dimerization motif, and the C-terminus of the MHC class II β chain or portion thereof is linked to a Jun leucine zipper dimerization motif. Optionally, the MHC class II α chain or portion thereof is linked to the Jun leucine zipper dimerization motif by an MHC-Jun linker, and the MHC class II β chain or portion thereof is linked to the Fos leucine zipper dimerization motif by an MHC-Fos linker.Optionally, the MHC class II α chain or portion thereof is linked to a Fos leucine zipper dimerization motif by an MHC-Fos linker, and the MHC class II β chain or portion thereof is linked to a Jun leucine zipper dimerization motif by an MHC-Jun linker. Optionally, the MHC-Jun linker and the MHC-Fos linker each comprise the sequence set forth in SEQ ID NO: 1.

[0008] In some such compositions, 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. In some such compositions, the MHC ligand peptide is 10 to 18 amino acids in length, 10 to 15 amino acids in length, or 10 to 12 amino acids in length. In some such compositions, the MHC ligand peptide comprises residues P-1 to P9 or residues P-3 to P9. In some such compositions, the MHC ligand peptide is an antigenic MHC ligand peptide. In some such compositions, the MHC ligand peptide is associated with a T-cell mediated disease.

[0009] In some such compositions, the peptide linker linking the MHC ligand peptide to the MHC class II molecule is a flexible linker. In some such compositions, the peptide linker linking 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 such compositions, the peptide linker linking the MHC ligand peptide to the MHC class II molecule does not comprise any charged amino acids. In some such compositions, the peptide linker linking the MHC ligand peptide to the MHC class II molecule comprises a cleavage site. Optionally, the cleavage site is a tobacco etch virus (TEV) protease cleavage site.

[0010] In some such compositions, the peptide linker linking the MHC ligand peptide to the MHC class II molecule is non-immunogenic. In some such compositions, the peptide linker linking 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 such compositions, the peptide linker linking 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 such compositions, the peptide linker linking the MHC ligand peptide to the MHC class II molecule is at least about 9 amino acids in length. In some such compositions, the peptide linker linking the MHC ligand peptide to the MHC class II molecule is at least 9 amino acids in length. In some such compositions, the peptide linker linking the MHC ligand peptide to the MHC class II molecule is about 9 to about 50 amino acids in length. In some such compositions, the peptide linker linking the MHC ligand peptide to the MHC class II molecule is 9 to 50 amino acids in length. In some such compositions, the peptide linker linking the MHC ligand peptide to the MHC class II molecule comprises 2 to 4 repeats of the sequence set forth in SEQ ID NO:4.

[0011] In some such compositions, the peptide linker linking 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 linking the MHC ligand peptide to the MHC class II molecule. Optionally, the first cysteine ​​is in the first four amino acids of the peptide linker linking the MHC ligand peptide to the MHC class II molecule. In some such compositions, the peptide linker linking the MHC ligand peptide to the MHC class II molecule comprises two to four repeats of the sequence set forth in SEQ ID NO:4, wherein one amino acid in one of the repeats is mutated to a cysteine. Optionally, the peptide linker linking the MHC ligand peptide to the MHC class II molecule comprises the sequence set forth in SEQ ID NO:21.

[0012] In some such compositions, the MHC ligand peptide comprises a first cysteine. Optionally, the first cysteine ​​faces away from the epitope formed by the composition.

[0013] In some such compositions, the second cysteine ​​is in an MHC class II α chain or portion thereof. Optionally, the peptide linker linking the MHC ligand peptide to the MHC class II molecule is attached to the N-terminus of an MHC class II β chain or portion thereof.

[0014] In some such compositions, the second cysteine ​​is not present in a wild-type MHC class II molecule corresponding to the MHC class II molecule in the composition. Optionally, the second cysteine ​​is present in place of a non-cysteine ​​amino acid in the corresponding wild-type MHC class II molecule. Optionally, the second cysteine ​​is in an MHC class II α chain or a portion thereof. Optionally, the second cysteine ​​is at a position corresponding to position 101 of the sequence set forth in SEQ ID NO:49 when the MHC class II α chain or a portion thereof is optimally aligned with SEQ ID NO:49. For example, the second cysteine ​​can be at a position corresponding to the position labeled DQA1R101 in the alignment of the full-length sequences of HLA-DPA1, HLA-DQA1, and HLA-DRA1 in Figure 3. For example, the second cysteine ​​in the corresponding wild-type MHC class II α chain can be at a position corresponding to position 78 of the sequence set forth in SEQ ID NO:59 when the MHC class II α chain or a portion thereof is optimally aligned with SEQ ID NO:59.

[0015] In some such compositions, the MHC class II molecules lack cysteines present in the corresponding wild-type MHC class II molecules. Optionally, the cysteines present in the corresponding wild-type MHC class II molecules are substituted with other amino acids. Optionally, the cysteines present in the corresponding wild-type MHC class II molecules are substituted with alanine, glutamine, tryptophan, or arginine. Optionally, the cysteines present in the corresponding wild-type MHC class II molecules are substituted with alanine or glutamine in the MHC class II molecules in the composition.

[0016] In some such compositions, the MHC class II α chain or portion thereof lacks a 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 is substituted with alanine or glutamine in the MHC class II α chain or portion thereof in the composition. Optionally, the cysteine ​​in the corresponding wild-type MHC class II α chain is at a position corresponding to position 70 of the sequence set forth in SEQ ID NO: 49 when the MHC class II α chain or portion thereof is optimally aligned with SEQ ID NO: 49. For example, the cysteine ​​in the corresponding wild-type MHC class II α chain can be at a position corresponding to the position designated DQA1C70 in the alignment of the full-length sequences of HLA-DPA1, HLA-DQA1, and HLA-DRA1 in Figure 3. For example, the cysteine ​​in the corresponding wild-type MHC class II α chain can be at a position corresponding to position 47 of the sequence set forth in SEQ ID NO: 59 when optimally aligned with the MHC class II α chain or portion thereof.

[0017] 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 an MHC class II α chain or a portion thereof and / or an MHC class II β chain or a portion thereof.

[0018] In some such 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.

[0019] In some such compositions, the MHC class II α chain or portion thereof comprises an MHC class II α chain extracellular domain, the MHC class II β chain or portion thereof comprises an MHC class II β chain extracellular domain, the peptide linker linking the MHC ligand peptide to the MHC class II molecule is a flexible linker about 9 to about 50 amino acids in length that includes a first cysteine ​​and is connected to the N-terminus of the MHC class II β chain or portion thereof, and a second cysteine ​​in the MHC class II α chain or portion thereof that is not present in a wild-type MHC class II molecule corresponding to the MHC class II molecule in the composition, and the MHC class II molecule lacks the cysteine ​​present in the corresponding wild-type MHC class II molecule. In some such compositions, the MHC class II α chain or portion thereof comprises an MHC class II α chain extracellular domain, the MHC class II β chain or portion thereof comprises an MHC class II β chain extracellular domain, the peptide linker linking the MHC ligand peptide to the MHC class II molecule is a flexible linker 9 to 50 amino acids in length that includes a first cysteine ​​and is connected to the N-terminus of the MHC class II β chain or portion thereof, and a second cysteine ​​in the MHC class II α chain or portion thereof that is not present in a wild-type MHC class II molecule corresponding to the MHC class II molecule in the composition, and the MHC class II molecule lacks the cysteine ​​present in the corresponding wild-type MHC class II molecule. Optionally, the composition is soluble, and the MHC class II α chain or portion thereof comprises an α1 domain and an α2 domain but does not comprise a transmembrane or cytoplasmic domain, and the MHC class II β chain or portion thereof comprises a β1-containing domain and a β2 domain but does not comprise a transmembrane or cytoplasmic domain, and the MHC class II α chain or portion thereof and the MHC class II β chain or portion thereof are linked by a Jun-Fos zipper comprising a Jun leucine zipper dimerization motif and a Fos leucine zipper dimerization motif.Optionally, the second cysteine ​​is at a position corresponding to position 101 of the sequence set forth in SEQ ID NO: 49 when the MHC class II α chain or portion thereof is optimally aligned with SEQ ID NO: 49, and a cysteine ​​in a corresponding wild-type MHC class II molecule is at a position corresponding to position 70 of the sequence set forth in SEQ ID NO: 49 when the MHC class II α chain or portion thereof is optimally aligned with SEQ ID NO: 49. For example, the second cysteine ​​can be at a position corresponding to the position designated DQA1 R101 in the alignment of the full-length sequences of HLA-DPA1, HLA-DQA1, and HLA-DRA1 in Figure 3, and a cysteine ​​in a corresponding wild-type MHC class II α chain can be at a position corresponding to the position designated DQA1 C70 in the alignment of the full-length sequences of HLA-DPA1, HLA-DQA1, and HLA-DRA1 in Figure 3. For example, the second cysteine ​​of the corresponding wild-type MHC class II α chain can be at a position corresponding to position 78 of the sequence set forth in SEQ ID NO: 59 when the MHC class II α chain or portion thereof is optimally aligned with SEQ ID NO: 59, and the cysteine ​​of the corresponding wild-type MHC class II α chain can be at a position corresponding to position 47 of the sequence set forth in SEQ ID NO: 59 when the MHC class II α chain or portion thereof is optimally aligned with 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.

[0020] Optionally, the MHC class II α chain extracellular domain comprises SEQ ID NO: 64. Optionally, the MHC class II α chain extracellular domain consists essentially of SEQ ID NO: 64. Optionally, the MHC class II α chain extracellular domain consists of SEQ ID NO: 64. Optionally, the MHC class II α chain or portion thereof (e.g., the MHC class II α chain extracellular domain) is linked to (e.g., at the C-terminus) 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 consists essentially of SEQ ID NO: 23. Optionally, the Fos leucine zipper dimerization motif consists essentially of SEQ ID NO: 23. Optionally, the MHC class II β chain extracellular domain comprises SEQ ID NO: 60. Optionally, the MHC class II β chain extracellular domain consists essentially of SEQ ID NO: 60. Optionally, the MHC class II beta chain extracellular domain consists of SEQ ID NO: 60. Optionally, the MHC class II beta chain or portion thereof (e.g., the MHC class II beta chain extracellular domain) is linked to (e.g., at the C-terminus) a Jun leucine zipper dimerization motif. Optionally, the Jun leucine zipper dimerization motif comprises SEQ ID NO: 24. Optionally, the Jun leucine zipper dimerization motif consists essentially of SEQ ID NO: 24. Optionally, the Jun leucine zipper dimerization motif consists of SEQ ID NO: 24. Optionally, the Jun leucine zipper dimerization motif is linked to the Jun leucine zipper dimerization motif by a linker. Optionally, the linker comprises SEQ ID NO: 1. Optionally, the linker consists essentially of SEQ ID NO: 1. Optionally, the linker consists of SEQ ID NO: 1. Optionally, the N-terminus of the MHC class II β chain or portion thereof (e.g., the MHC class II β chain extracellular domain) is linked to the MHC ligand peptide (e.g., the C-terminus of the MHC ligand peptide) by 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, or 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, or 10 to 15 amino acids in length, or 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.

[0021] In another aspect, a nucleic acid encoding any of the above compositions is provided.

[0022] In some aspects, methods of eliciting an immune response in a subject are provided. Some such methods include administering to the subject an effective amount of any of the compositions described above, or a nucleic acid encoding the composition.

[0023] In another aspect, methods of producing antigen binding proteins are provided. Some such methods comprise: (a) immunizing a non-human animal with any of the compositions described above or a nucleic acid encoding the composition; and (b) maintaining the non-human animal under conditions sufficient for the non-human animal to mount an immune response to the composition. Optionally, the antigen binding protein specifically binds to an antigen composition comprising an MHC ligand peptide covalently linked 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 of producing antigen binding proteins specifically bind to an antigenic composition comprising an MHC ligand peptide covalently linked to an MHC class II molecule are provided. Some such methods comprise: (a) immunizing a non-human animal with any of the compositions described above or a nucleic acid encoding the composition; and (b) maintaining the non-human animal under conditions sufficient for the non-human animal to mount 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. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 (not to scale) shows several embodiments of various soluble peptide-MHC II constructs. The sequence of the linker (SGGGGG) used in some constructs to link the Fos and Jun leucine zipper dimerization motifs to the α and β chains is set forth in SEQ ID NO: 1. Labels indicate the cysteines incorporated into the linker (LinkerCys) and α chain (R101C) of construct B for disulfide stapling of the peptides. Labels also indicate that the cysteine ​​at position 70 of the α chain is mutated to glutamine (C70Q) or alanine (C70A). Asterisks indicate Davis-body modifications (CH3 modifications that allow differential binding of Fc to Protein A). [Figure 2]Alignment of full-length DQ2 α-chain segments containing no mutation, the C70Q mutation, or the R101C and C70A mutations is shown. [Figure 3] An alignment of full-length α chain segments from different HLA class II alleles is shown. [Figure 4] 1 shows results from a Biacore assay demonstrating that soluble construct C in some embodiments binds to an anti-class II monoclonal antibody captured on an anti-mFc sensor surface. [Figure 5] 1 shows results from a Biacore assay demonstrating that soluble construct C captured on an anti-hFc sensor surface binds to an anti-class II monoclonal antibody in some embodiments. [Figure 6] In some embodiments, soluble constructs are shown in which peptides are tethered to HLA-DQB chains and HLA α and β chains are dimerized in either Jun / Fos or Fc knobs-into-holes configurations. DETAILED DESCRIPTION OF THE INVENTION

[0025] definition The terms "protein," "polypeptide," and "peptide," used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and amino acids that are chemically or biochemically modified or derivatized. These terms also include modified polymers, such as polypeptides with modified peptide backbones. The term "domain" refers to any portion of a protein or polypeptide having a specific function or structure.

[0026] Proteins are said to have an "N-terminus" (amino terminus) and a "C-terminus" (carboxy or carboxyl terminus). The term "N-terminus" refers to the beginning of a protein or polypeptide, which ends with an amino acid having a free amine group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH).

[0027] The terms "nucleic acid" and "polynucleotide," used interchangeably herein, include polymeric forms of nucleotides of any length, containing ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. These include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers that contain purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0028] Nucleic acids are said to have a "5' end" and a "3' end" because mononucleotides react to form oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is unidirectionally linked to the 3' oxygen of its neighbor via a phosphodiester bond. An end of an oligonucleotide is called the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is called the "3' end" if its 3' oxygen is not linked to the 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence can also be said to have 5' and 3' ends, even if it is internal to a larger oligonucleotide. In either a linear or circular DNA molecule, distinct elements are referred to as "upstream" or "downstream" 5' or 3' elements.

[0029] The terms "expression vector" or "expression construct" or "expression cassette" refer to a recombinant nucleic acid comprising a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host cell or organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), a ribosome binding site, and other sequences. Eukaryotic cells are known to generally utilize promoters, enhancers, termination and polyadenylation signals, and some elements can be deleted and others added without sacrificing the required expression.

[0030] A "promoter" is a regulatory region of DNA that usually contains a TATA box capable of directing RNA polymerase II to begin RNA synthesis at the appropriate transcription start site for a particular polynucleotide sequence.

[0031] In some embodiments of the present invention, the promoter may further comprise other regions that affect the rate of transcription initiation. In some embodiments, the promoter sequences disclosed herein regulate the transcription of an operably linked polynucleotide. The promoter may be active in one or more cell types disclosed herein (e.g., but not limited to, eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or a combination thereof). The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., but not limited to, a developmentally regulated promoter), or a spatially restricted promoter (e.g., but not limited to, a cell-specific or tissue-specific promoter).

[0032] "Operable linkage" or "operably linked" includes the proximity of two or more components (for example, but not limited to, a promoter and another sequence element) such that both components function normally and at least one of the components can mediate the function of at least one of the other components. As a non-limiting example, a promoter can be operably linked to a coding sequence if it controls the level of transcription of the coding sequence depending on the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences being in close proximity to each other or acting in trans (for example, but not limited to, regulatory sequences can act at a distance to control transcription of the coding sequence).

[0033] The term "isolated" with respect to proteins, nucleic acids, and cells includes proteins, nucleic acids, and cells that are relatively purified with respect to other cellular or biological components that may normally be present in situ, up to and including substantially pure preparations of proteins, nucleic acids, or cells.

[0034] In some embodiments of the invention, the term "isolated" encompasses proteins and nucleic acids that have no naturally occurring counterpart, or proteins or nucleic acids that are chemically synthesized and thus are substantially free from contaminating other proteins or nucleic acids. The term "isolated" can also include proteins, nucleic acids, or cells that have been separated or purified from most other cellular or biological components with which they are naturally associated (e.g., other cellular proteins, nucleic acids, or cellular or extracellular components).

[0035] "Codon optimization" refers to the process of modifying a nucleic acid sequence to enhance expression in a particular host cell by taking advantage of codon degeneracy, as indicated by the diversity of three-base pair codon combinations that specify amino acids, and generally by replacing at least one codon of the native sequence with a codon more frequently or most frequently used in the host cell's genes while maintaining the native amino acid sequence. As a non-limiting example, a nucleic acid encoding a protein can be modified to use alternative codons that are more frequently used 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 adapted in various ways. See Nakamura et al. (2000) Nucleic Acids Res. 28(1):292, incorporated herein by reference in its entirety for all purposes. Computer algorithms are also available for codon optimization of a particular sequence for expression in a particular host (see, eg, Gene Forge).

[0036] The term "locus" refers to the specific location of a gene (or key sequence), DNA sequence, polypeptide-coding sequence, or position on a chromosome in the genome of an organism. As a non-limiting example, an "HLA locus" can refer to the specific location of an HLA gene, an HLA DNA sequence, a sequence encoding an HLA, or the location of an HLA on a chromosome in the genome of an organism in which such a sequence is identified as residing. An "HLA locus" can include regulatory elements of an HLA gene, including, as non-limiting examples, an enhancer, a promoter, a 5' and / or a 3' untranslated region (UTR), or a combination thereof.

[0037] The term "gene" refers to a DNA sequence in a chromosome that, when present in nature, may contain at least one coding region and at least one non-coding region. A DNA sequence in a chromosome that encodes a product (e.g., but not limited to, an RNA product and / or a polypeptide product) may include coding regions interrupted by non-coding introns and sequences located adjacent to the coding region at both the 5' and 3' ends, such that the gene corresponds to a full-length mRNA (including 5' and 3' untranslated sequences). In addition, 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 also be present in a gene. These sequences may be adjacent (e.g., within 10 kb) or distant from the coding region of the gene, and they affect the level or rate of gene transcription and translation.

[0038] The term "allele" refers to variant forms of a gene. Some genes have different forms that are located at the same position, or locus, on a chromosome. Diploid organisms have two alleles at each locus. Each pair of alleles represents a genotype at a particular locus. A genotype is described as homozygous if there are two identical alleles at a particular locus, and as heterozygous if the two alleles are different.

[0039] The methods and compositions provided herein use a variety of different components. Some components throughout the description may have active variants and fragments. Such components include, for example, MHC class II molecules. The biological activities of each of these components are described elsewhere herein. The term "functional" refers to the inherent ability of a protein or nucleic acid (or a fragment or variant thereof) to exhibit a biological activity or function. Such biological activity or function can include, for example, the ability of an MHC class II molecule to bind to an MHC ligand peptide and / or to bind to a T cell receptor (TCR) and produce a T cell response. The biological function of a functional fragment or variant may be the same or may actually change compared to the original molecule (e.g., without limitation, with respect to their specificity, selectivity, or efficacy), while retaining the basic biological function of the molecule.

[0040] The term "wild-type" refers to an entity having a structure (e.g., without limitation, a nucleic acid sequence or an amino acid sequence) as found in a normal state or context (as opposed to mutant, diseased, altered, etc.). Wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0041] The term "variant" refers to a nucleotide sequence (for example, but not limited to, by one nucleotide) that differs from the most common sequence in a population, or a protein sequence (for example, but not limited to, by one amino acid) that differs from the most common sequence in a population.

[0042] The term "fragment," when referring to a protein, refers to a protein that is shorter or has fewer amino acids than the full-length protein. The term "fragment," when referring to a nucleic acid, refers to a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. Non-limiting examples of protein fragments can include N-terminal fragments (i.e., removal of a portion of the C-terminus of the protein), C-terminal fragments (i.e., removal of a portion of the N-terminus of the protein), or internal fragments (i.e., removal of a portion of an internal portion of the protein).

[0043] "Sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues of the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When using percentage sequence identity in proteins, non-identical residue positions often differ by conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue that has similar chemical properties (e.g., but not limited to, charge or hydrophobicity) and therefore does not alter the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, as a non-limiting example, conservative substitutions are given a score of zero to 1, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of zero. Scoring of conservative substitutions is calculated, for example, as performed in the program PC / GENE (Intelligenetics, Mountain View, California).

[0044] "Percentage of sequence identity" includes a value determined by comparing two optimally aligned sequences (maximum number of perfectly matched residues) over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) when compared to a reference sequence (not including additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a concatenated non-homologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0045] Unless otherwise specified, sequence identity / similarity values ​​include values ​​obtained using GAP version 10 with the following parameters: % identity and % similarity for nucleotide sequences using a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program. "Equivalent program" includes any sequence comparison program that produces alignments with identical nucleotide or amino acid residue matches and identical percent sequence identity for any two sequences in question when compared to corresponding alignments produced by GAP version 10.

[0046] The term "in vitro" includes an artificial environment and processes or reactions that occur within an artificial environment (e.g., without limitation, a test tube or an isolated cell or cell line). The term "in vivo" refers to a natural environment (e.g., without limitation, an organism or body or a cell or tissue within an organism or body) and processes or reactions that occur within a natural environment. The term "ex vivo" includes cells removed from an individual's body and processes or reactions that occur within such cells.

[0047] The terms "major histocompatibility complex" and "MHC" encompass "human leukocyte antigen" or "HLA" (the latter two being commonly used 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, the α1, α2, and / or α3 subunits of the MHC class I α chain, the α1-α2 subunits of the MHC class II α chain, the β1-β2 subunits of the MHC class II β chain), as well as portions (e.g., but not limited to, peptide-binding portions such as peptide-binding grooves), variants, and various derivatives (including fusion proteins) thereof, which portions, variants, and derivatives retain the ability to present antigenic peptides for recognition by a T cell receptor (TCR) (e.g., but not limited to, an antigen-specific TCR). MHC class I molecules contain a peptide-binding groove formed by the α1 and α2 domains of the heavy α chain, which can accommodate peptides of approximately 8-10 amino acids. Despite the fact that MHC of either class binds to a core of approximately 9 amino acids (e.g., 5-17 amino acids) within a peptide, the open-ended nature of the MHC class II peptide-binding groove (the α1 domain of a class II MHC α polypeptide associates with the β1 domain of a class II MHC β polypeptide) allows for a wider range of peptide lengths. Peptides that bind to MHC class II typically range in length from 13-17 amino acids, but can also be shorter or longer. As a result, peptides shift within the MHC class II peptide-binding groove, potentially changing which 9-mer sequence is directly positioned within the groove at any given time. Conventional methods for identifying specific MHC variants are used herein.

[0048] The term "antigen" refers to any agent (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portion thereof, or combination thereof) that, when introduced into an immunocompetent host, is recognized by the host's immune system and elicits an immune response by the host. TCR recognizes peptides presented in the context of MHC as part of the immune synapse. Peptide-MHC (pMHC) complexes are recognized by TCRs, with the peptide (antigenic determinant) and TCR idiotype providing the specificity of the interaction. Thus, the term "antigen" encompasses, but is not limited to, peptides presented in the context of MHC (e.g., peptide-MHC complexes or pMHC complexes). Peptides presented on MHC are sometimes referred to as "epitopes" or "antigenic determinants." Terms such as "peptide," "antigenic determinant," and "epitope" encompass not only those naturally presented by antigen-presenting cells (APCs), but also any desired peptide recognized by immune cells (e.g., but not limited to, when properly presented to cells of the immune system).

[0049] "Peptide-MHC class II complex," "pMHC class II complex," "peptide in groove," and the like include (i) an MHC class II molecule (e.g., but not limited to, a human MHC class II molecule) or a portion thereof (e.g., its peptide-binding groove or an extracellular portion thereof), and (ii) an antigenic peptide, wherein the MHC class II molecule and the antigenic peptide form a complex such that the pMHC class II complex specifically binds to a T cell receptor. pMHC class II complexes include pMHC class II complexes expressed on the cell surface and soluble pMHC class II complexes. By administering to an animal an antigenic pMHC class II complex (e.g., but not limited to, a pMHC class II complex complexed with a peptide (e.g., a peptide that is foreign to the animal receiving the complex containing the MHC class II molecule)), the animal can generate an antibody response to the antigenic pMHC class II complex and / or generate a T cell response to the antigenic pMHC class II complex (i.e., generate a T cell receptor specific for the pMHC class II complex). Such specific antigen-binding proteins can then be isolated and used as therapeutic agents to specifically modulate specific T cell receptor interactions with antigenic pMHC class II complexes. In some cases, soluble pMHC class II complexes containing peptides complexed with MHC class II molecules (e.g., but not limited to, peptides that are foreign to the host animal to which the pMHC class II complexes are administered) may not elicit a T cell immune response, depending on the soluble nature of the administered pMHC class II complex, but such soluble pMHC class II complexes may still be considered antigenic in that they can elicit a B cell-mediated immune response that generates an antigen-binding protein that specifically binds to the soluble pMHC class II complex.

[0050] The term "effective amount" encompasses an amount effective to achieve a desired result, at the dosage and for the duration necessary. The effective amount of a peptide-MHC class II complex may vary depending on factors such as the subject's condition, age, and weight, and the ability of the peptide-MHC class II complex to elicit a desired response in the subject. The administration regimen may be adjusted to provide an optimal response. An effective amount is also an amount in which the therapeutically beneficial effects outweigh the toxic or harmful effects (e.g., including but not limited to, side effects) of the peptide-MHC class II complex.

[0051] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes examples when the event or circumstance occurs and examples when it does not occur.

[0052] The specification of a range of values ​​includes all integers within or defining the range, and all subranges defined by integers within the range.

[0053] Unless otherwise clear from the context, the term "about" encompasses values ​​that are ±5 of the stated value.

[0054] The term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0055] The term "or" refers to any one member of a particular list and also includes any combination of members of that list.

[0056] The singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "protein" or "at least one protein" can include a plurality of proteins, including mixtures thereof.

[0057] Statistically significant means p≦0.05. (Mode for Carrying Out the Invention)

[0058] I. Overview Provided herein are compositions comprising an MHC ligand peptide covalently linked to an MHC class II molecule. In some embodiments of the present invention, the MHC class II molecule can 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 composition embodiments, the MHC ligand peptide is covalently linked to the MHC class II molecule by a peptide linker. The MHC ligand peptide or peptide linker can comprise a first cysteine, 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, can comprise a second cysteine. In some embodiments, the first cysteine ​​and the second cysteine ​​can then form a disulfide bond such that the MHC ligand peptide binds to the 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 eliciting an immune response in a subject using such compositions are also provided.

[0059] Soluble peptide-MHC I protein constructs have been previously described. These constructs can be used for a variety of applications, including immunizing rodents (such as VELOCIMMUNE® rodents) to generate anti-peptide-in-groove antibodies or for generating T cell receptors specific for peptide-MHC I proteins. The inventors have designed peptide-MHC II protein constructs in which the α and β chains of an MHC II molecule are locked together and anchored to a peptide in its groove. These can be used for a variety of applications, including, but not limited to, generating soluble MHC II constructs that function as immunogens, as well as generating membrane-anchored MHC II proteins for other applications, including recruitment of T cells expressing MHC class II peptide-specific TCRs.

[0060] II. Compositions Comprising Peptide-MHC Class II Complexes In some embodiments of the present 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 linked 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 complex embodiments, the MHC ligand peptide is covalently linked to the MHC class II molecule by a peptide linker. The MHC ligand peptide or peptide linker can include a first cysteine, and the MHC class II molecule (e.g., 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) can include a second cysteine, and the first and second cysteines form a disulfide bond such that the MHC ligand peptide binds in the 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.

[0061] In some embodiments, an MHC class II molecule useful as part of an antigenic peptide-MHC class II complex can comprise at least a portion, fragment, or variant of an MHC class II α chain and at least a portion, fragment, or variant of an MHC class II β chain (e.g., at least a portion, fragment, or variant of the extracellular domain of an MHC class II α chain and at least a portion, fragment, or variant of the extracellular domain of an MHC class II β chain), such that the portion, fragment, or variant of the MHC class II α chain and the portion, fragment, or variant of the MHC class II β chain form a peptide-binding groove capable of binding to an MHC ligand peptide. By way of non-limiting example, MHC class II molecules useful as part of an antigenic peptide-MHC class II complex can include naturally occurring full-length MHC, as well as 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 subunit of the MHC class II α chain and the β1-β2 subunit 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), and fragments, variants, and derivatives thereof (including fusion proteins), which retain the ability to present antigenic determinants for recognition by an antigen-specific T cell receptor (TCR). MHC class II molecules and MHC class II molecules useful as part of an antigenic peptide-MHC class II complex are described in more detail elsewhere herein.

[0062] In some embodiments of the complex, at least one chain of an MHC class II molecule (e.g., 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 as a fusion protein. As a non-limiting example, the MHC class II molecule (e.g., 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 can be connected via a linker. Linking of an MHC class II molecule to an MHC ligand peptide and suitable linkers therefor are described in more detail below.

[0063] In some embodiments of the complex, the MHC ligand peptide or the linker connecting the MHC ligand peptide to at least one chain of the MHC class II molecule (or a portion or fragment or variant thereof) is bonded via a disulfide bridge. A disulfide bridge is a disulfide bond extending between a pair of oxidized cysteines. The attachment of the MHC ligand peptide or the linker connecting the MHC ligand peptide to at least one chain of the MHC class II molecule (or a portion or fragment or variant thereof) via a disulfide bridge is described in more detail below.

[0064] The peptide-MHC class II complexes disclosed in some embodiments herein can be membrane-bound or soluble. MHC class II molecules are naturally membrane-anchored heterodimers. The hydrophobic transmembrane regions of the α and β chains promote heterodimer assembly. Some of the peptide-MHC class II complexes herein are membrane-bound. As a non-limiting example, such membrane-bound peptide-MHC class II complexes can include MHC class II molecules that contain a transmembrane domain or contain a transmembrane domain and a cytoplasmic domain. As a non-limiting example, the MHC class II molecule in the complex can contain an α chain that contains a transmembrane domain, or contain a transmembrane domain and a cytoplasmic domain, and / or the MHC class II molecule can contain a β chain that contains a transmembrane domain, or contain a transmembrane domain and a cytoplasmic domain.

[0065] In some embodiments, the peptide-MHC class II complex can be soluble (i.e., not membrane-bound). As a non-limiting example, such a soluble peptide-MHC class II complex can comprise an MHC class II molecule that does not contain a transmembrane domain or that does not contain a transmembrane domain and a cytoplasmic domain. As a non-limiting example, the MHC class II molecule in the complex can comprise an α chain that does not contain a transmembrane domain or that does not contain a transmembrane domain and a cytoplasmic domain, and / or the MHC class II molecule can comprise a β chain that does not contain a transmembrane domain or that does not contain a transmembrane domain and a cytoplasmic domain.

[0066] In some embodiments, the soluble peptide-MHC class II complex can further comprise other components for stabilizing chain pairing 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. In some embodiments, non-limiting examples of mechanisms for stabilizing chain pairing include binding to a Jun-Fos zipper, binding to an immunoglobulin scaffold, binding to an immunoglobulin Fc region (e.g., an immunoglobulin Fc hinge region), electrostatic engineering such as immunoglobulin Fc knob-into-hole mutations, immunoglobulin Fc charge mutations (including, but not limited to, charge reversal mutations), direct linkers (e.g., 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, other means suitable for chain pairing can also be used.

[0067] A. MHC class II molecules 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 broadly classified into two categories: class I and class II MHC molecules. MHC class II molecules or proteins are heterodimeric integral membrane proteins comprising one α chain and one β chain in non-covalent interactions. The α chain has two extracellular domains (α1 and α2) and two extracellular domains (TM and CYT domains). The β chain has two extracellular domains (β1 and β2) and two extracellular domains (TM and CYT domains).

[0068] The domain organization of class II MHC molecules forms the antigenic determinant binding site (e.g., peptide-binding portion or peptide-binding groove) of the MHC molecule. The peptide-binding groove refers to the portion of the MHC protein that forms a cavity to which a peptide (e.g., an antigenic determinant) can bind. The conformation of the peptide-binding groove changes upon peptide binding, allowing for proper alignment of amino acid residues important for TCR binding to the peptide-MHC (pMHC) complex.

[0069] In some embodiments of peptide-MHC class II complexes, the MHC class II molecule comprises a portion, fragment, or variant of a class II MHC chain sufficient to form a peptide-binding groove. The peptide-binding groove of a class II protein can comprise a portion, fragment, or variant of the α1 and β1 domains capable of forming two β-pleated sheets and two α-helices. The first portion of the α1 domain forms the first β-pleated sheet, and the second portion of the α1 domain forms the first α-helix. The first portion of the β1 domain forms the second β-pleated sheet, and the second portion of the β1 domain forms the second α-helix. X-ray crystal structures of class II proteins with peptides engaged in the protein's binding groove indicate that one or both ends of the engaged peptide may protrude beyond the MHC protein. See, e.g., Brown et al. (1993) Nature 364(6432):33-39, incorporated herein by reference in its entirety for all purposes. Thus, the ends of the class II α1 and β1 α-helices form an open cavity such that the end of the peptide bound in the binding groove is not buried in the cavity.

[0070] Many human and mammalian MHCs are known. As a non-limiting example of some embodiments, human MHC II α or β polypeptides can be derived from the α or β polypeptides of functional human HLA molecules encoded by any of the HLA-DP, HLA-DQ, HLA-DR, HLA-DM, and HLA-DO loci, or a combination thereof. A list of commonly used HLA antigens and alleles, as well as a brief description of HLA nomenclature, is outlined in Shankarkumar et al., "The Human Leukocyte Antigen (HLA) System," Int. J. Hum. Genet. 4(2):91-103, (2004), which is incorporated herein by reference in its entirety for all purposes. Additional information regarding HLA nomenclature and various HLA alleles can be found in Holdsworth et al. (2009) Tissue Antigens 73(2):95-170 and Marsh (2019) Int. J. Immunogenet. 46(5):346-418, each of which is incorporated by reference in its entirety for all purposes.

[0071] In one exemplary embodiment, the MHC is a human MHC class II molecule, such as a cell surface-expressed human HLA molecule 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 can include one or more MHC class II α chains or domains, or portions, fragments, or variants thereof (e.g., without limitation, one or more human MHC class II α chains, or domains, portions, fragments, or variants thereof). As a non-limiting exemplary embodiment, the class II α chain can be HLA-DPA, HLA-DQA, or HLA-DRA. Similarly, in some embodiments, the peptide-MHC class II complex can include one or more MHC class II β chains, or domains, portions, fragments, or variants thereof (e.g., without limitation, one or more human MHC class II β chains, or domains, portions, fragments, or variants thereof). As a non-limiting exemplary embodiment, the class II β chain can be HLA-DPB, HLA-DQB, or HLA-DRB.

[0072] In some embodiments, of particular interest are polymorphic human HLA alleles known to be associated with a number of human diseases, including, but not limited to, human autoimmune diseases. Specific polymorphisms at HLA loci have been identified that correlate with the development of rheumatoid arthritis, type I diabetes, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, Graves' disease, systemic lupus erythematosus, celiac disease, Crohn's disease, ulcerative colitis, and other autoimmune diseases. See, e.g., de Bakker (2006) Nat. Genet. 38(10):1166-1172, Wong and Wen (2004) Diabetologia 47(9):1476-1487, Taneja and David (1998) J. Clin. Invest. 101(5):921-926, and International MHC and Autoimmunity Genetics Network (2009) Proc. Natl. Acad. Sci. USA 106(44):18680-18685, each of which is incorporated by reference in its entirety for all purposes. In some embodiments, the human MHC II polypeptide may be derived from a human HLA molecule known to be associated with a particular disease (such as, but not limited to, an autoimmune disease).

[0073] In one exemplary embodiment, the human MHC class II molecule (for example, but not limited to, human MHC II α and β polypeptides, or portions, fragments, or variants thereof) is derived from human HLA-DR (for example, but not limited to, HLA-DR2). Typically, the HLA-DR α chain is monomorphic (e.g., the α chain of the HLA-DR protein is encoded by an HLA-DRA gene, such as the HLA-DRα*01 gene). Meanwhile, the HLA-DR β 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. Any suitable HLA-DR sequence is encompassed herein, including polymorphic variants exhibited in the human population, sequences with one or more conservative or non-conservative amino acid modifications, etc.

[0074] In another exemplary embodiment, the human MHC class II molecule (for example, but not limited to, human MHC II α and β polypeptides, or portions, fragments, or variants thereof) is derived from human HLA-DQ (for example, but not limited to, HLA-DQ2). HLA-DQ2 is a serotype group determined by antibody recognition of the β2 subset of DQ β chains. The DQ β chain is encoded by the HLA-DQB1 locus, and DQ2 is encoded by the HLA-DQB1*02 allele group. This group includes two common alleles, DQB1*0201 and DQB1*0202. The DQ2 β chain combines with an α chain encoded by a genetically linked HLA-DQA1 allele to form a cis-haplotype isoform. These isoforms are called DQ2.2 and DQ2.5 and are encoded by the DQA1*0201 and DQA1*0501 genes, respectively. DQ2.5 is one of the most prominent causative factors of autoimmune diseases. DQ2.5 is often encoded by a haplotype associated with numerous diseases, including autoimmune diseases. This haplotype, HLA A1-B8-DR3-DQ2, is associated with diseases suspected to involve HLA-DQ2. For example, DQ2 is directly involved in celiac disease.

[0075] In another embodiment, the human MHC class II molecule (e.g., human MHC II α and β polypeptides, or portions, fragments, or variants thereof) can be encoded by the nucleotide sequence, or portions or fragments, of an HLA allele known to be associated with a common human disease, including, but 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) Nat. Genet. 38(10):1166-1172, which is incorporated herein by reference in its entirety for all purposes. Further non-limiting examples of HLA alleles associated with common diseases include B*0801 / DRB1*0301 / DQA1*0501 / DQB1*0201 (Graves' disease or severe lupus erythematosus), 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).

[0076] In some embodiments, an MHC class II molecule useful as part of an antigenic peptide-MHC class II complex comprises 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 (e.g., the extracellular domain of the MHC class II α chain, or a portion, fragment, or variant thereof, and the extracellular domain of the MHC class II β chain, or a portion, fragment, or variant thereof), wherein the MHC class II α chain and β chain (or a portion, fragment, or variant thereof) form a peptide-binding groove capable of binding to an MHC ligand peptide. By way of non-limiting example, MHC class II molecules useful as part of an antigenic peptide-MHC class II complex include naturally occurring full-length MHC as well as 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., the α1-α2 subunit of the MHC class II α chain and the β1-β2 subunit of the MHC class II β chain, or the MHC class II α chain and the β1 subunit of the MHC class II β chain), as well as portions, fragments, variants, and various derivatives thereof (including fusion proteins), which retain the ability to present antigenic determinants for recognition by an antigen-specific TCR. In one exemplary embodiment, the MHC class II α chain or portion, fragment, or variant thereof and the MHC class II β chain or portion, fragment, or variant thereof included in the peptide-MHC class II complex comprise the region or the minimum region required to form the peptide-binding groove of an MHC ligand peptide. In an exemplary embodiment, the MHC class II α chain or a part, fragment, or variant thereof and the MHC class II β chain or a part, fragment, or variant thereof contained in the peptide-MHC class II complex consist of the regions essentially required or the minimum required regions for forming the peptide-binding groove of the MHC ligand peptide.In an exemplary embodiment, the MHC class II α chain or a part, fragment, or variant thereof and the MHC class II β chain or a part, fragment, or variant thereof contained in the peptide-MHC class II complex consist of the region or the minimum region required to form the peptide-binding groove of the MHC ligand peptide.

[0077] The MHC class II molecules in the peptide-MHC complexes disclosed in some embodiments herein can be membrane-bound or soluble. MHC class II molecules are naturally membrane-anchored heterodimers. The hydrophobic transmembrane regions of the α and β chains promote heterodimer assembly. Some of the MHC class II molecules in the peptide-MHC class II complexes disclosed in some embodiments herein are membrane-bound. As a non-limiting example, such membrane-bound peptide-MHC class II complexes can include MHC class II molecules that include a transmembrane domain or a portion, fragment, or variant thereof, or that include a transmembrane and cytoplasmic domain or a portion, fragment, or variant thereof. As a non-limiting example, the MHC class II molecules in the complex can include an α chain that includes a transmembrane domain or a portion, fragment, or variant thereof, or that includes a transmembrane domain and a cytoplasmic domain or a portion, fragment, or variant thereof, and / or the MHC class II molecules can include a β chain that includes a transmembrane domain or a portion, fragment, or variant thereof, or that includes a transmembrane domain and a cytoplasmic domain or a portion, fragment, or variant thereof.

[0078] In some embodiments, the MHC class II molecule in the peptide-MHC class II complexes disclosed in some embodiments herein can be soluble (i.e., not membrane-bound). In exemplary embodiments, such soluble peptide-MHC class II complexes can comprise MHC class II molecules that do not contain a transmembrane domain or that do not contain a transmembrane domain and a cytoplasmic domain. In another exemplary embodiment, the MHC class II molecule in the complex can comprise an α chain or a portion, fragment, or variant thereof that does not contain a transmembrane domain or that does not contain a transmembrane domain and a cytoplasmic domain, and / or the MHC class II molecule can comprise a β chain or a portion, fragment, or variant thereof that does not contain a transmembrane domain or that does not contain a transmembrane domain and a cytoplasmic domain.

[0079] In one embodiment, the alpha chain or portion or fragment or variant thereof comprises a fragment of the full-length alpha chain that does not include the transmembrane domain or the C-terminal region of the C-terminal transmembrane domain. In another embodiment, the alpha chain or portion or fragment or variant thereof comprises a fragment of the full-length alpha chain that does not include a signal peptide at the N-terminus and does not include the transmembrane domain or the C-terminal region of the C-terminal transmembrane domain. In a non-limiting example, the alpha chain or portion or fragment or variant thereof can be operably linked to different signal peptides. In exemplary embodiments, the α chain, or 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 from which the α chain, or portion or fragment or variant thereof, is derived is optimally 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 portion or fragment or variant thereof, comprises amino acid residues 29-222 of SEQ ID NO:51, or a fragment 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 from which the α chain, or portion or fragment or variant thereof, is derived is optimally aligned with SEQ ID NO:51). In another embodiment, the alpha chain, or portion or fragment or variant thereof, comprises amino acid residues 26-216 of SEQ ID NO: 52 or 58, or a fragment of the full length alpha chain corresponding to amino acid residues 26-216 of SEQ ID NO: 52 or 58 (e.g., when the full length alpha chain from which the alpha chain, or portion or fragment or variant thereof, is derived is optimally aligned with SEQ ID NO: 52 or 58). In another embodiment, the alpha chain, or portion or fragment or variant thereof, comprises the sequence set forth in any one of SEQ ID NOs: 59 and 61-68.

[0080] In one embodiment, the β chain, or portion, fragment, or variant thereof, comprises a fragment of a full-length β chain that does not include the transmembrane domain or the C-terminal region of the C-terminal transmembrane domain. In another embodiment, the β chain, or portion, fragment, or variant thereof, comprises a fragment of a full-length β chain that does not include a signal peptide at the N-terminus and does not include the transmembrane domain or the C-terminal region of the C-terminal transmembrane domain. In a non-limiting example, the β chain, or portion, fragment, or variant thereof, can be operably linked to a different signal peptide. In an exemplary embodiment, the β chain, or portion, fragment, or variant thereof, comprises amino acid residues 33-230 of SEQ ID NO:50, or a fragment of a full-length β chain corresponding to amino acid residues 33-230 of SEQ ID NO:50 (e.g., when the full-length β chain from which the β chain, or portion, fragment, or variant thereof, is derived is optimally aligned with SEQ ID NO:50). In another exemplary embodiment, the β chain, or portion, fragment, or variant thereof, comprises the sequence set forth in SEQ ID NO:60.

[0081] In some embodiments, the MHC II molecule in the soluble peptide-MHC class II complex can further comprise other components for stabilizing the chain pairing 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. In some embodiments, non-limiting examples of mechanisms for stabilizing the chain pairing include binding to a Jun-Fos zipper, binding to an immunoglobulin scaffold, binding to an immunoglobulin Fc region (e.g., an immunoglobulin Fc hinge region), electrostatic engineering such as immunoglobulin Fc knob-into-hole, immunoglobulin Fc charge mutations (including, but not limited to, charge reversal mutations), direct linkers (e.g., covalent bonds such as peptide linkers), or any combination thereof. However, other means suitable for chain pairing can also be used.

[0082] In one 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 by a Jun-Fos zipper. Synthetic peptides of Fos and Jun leucine zipper dimerization motifs are known to assemble into stable, soluble heterodimers. See, for example, Kalandadze et al. (1996) J. Biol. Chem. 271:20156-20162 and Gauthier et al. (1998) Proc. Natl. Acad. Sci. USA 95:11828-11833, each of which is incorporated herein by reference in its entirety for all purposes. The leucine zipper is characterized by five leucines periodically arranged every seven residues (heptad repeats). Each heptad repeat contributes to the formation of two turns of an α-helix. Leucine residues have a special function in leucine zipper dimerization and form the interface between the two α-helices of the coiled-coil. The Jun / Fos heterodimer is soluble due to the presence of charged residues on the outer surface of the coiled-coil. 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) can be linked to the leucine zipper dimerization motifs derived from the transcription factors Fos and Jun, assembled into a soluble, compact coiled-coil 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 the leucine zipper dimerization motifs derived from the transcription factors Fos and Jun.In yet another exemplary embodiment, the extracellular domain of an MHC class II α chain, or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II α1 domain, or a portion or fragment or variant thereof, or an MHC class II α1 and α2 domains, or portions or fragments or variants thereof) can be linked (e.g., including but not limited to, fused in frame or fused via a linker) to the extracellular domain of a Fos leucine zipper dimerization motif, and the extracellular domain of an MHC class II β chain, or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II β1 domain, or a portion or fragment or variant thereof, or an MHC class II β1 and β2 domains, or portions or fragments or variants thereof) can be linked (e.g., including but not limited to, fused in frame or fused via a linker) to a Jun leucine zipper dimerization motif. In another exemplary embodiment, the extracellular domain of an MHC class II α chain, or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II α1 domain, or a portion or fragment or variant thereof, or an MHC class II α1 and α2 domains, or portions or fragments or variants thereof) can be linked (e.g., including but not limited to, fused in frame or fused via a linker) to the extracellular domain of a Jun leucine zipper dimerization motif, and the extracellular domain of an MHC class II β chain, or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II β1 domain, or a portion or fragment or variant thereof, or an MHC class II β1 and β2 domains, or portions or fragments or variants thereof) can be linked (e.g., including but not limited to, fused in frame or fused via a linker) to a Fos leucine zipper dimerization motif.The linkage (for example, without limitation, fusion in frame or fusion via a linker) can be, for example, at the C-terminus of the extracellular domain of the MHC class II α chain, or a portion, fragment, or variant thereof, and the C-terminus of the extracellular domain of the MHC class II β chain, or a portion, fragment, or variant thereof. Suitable linkers for linking the Jun leucine zipper dimerization motif and / or the Fos leucine zipper dimerization motif to an MHC class II molecule are disclosed in more detail elsewhere herein. Optionally, in a non-limiting example, the linker comprises SGGGGG (SEQ ID NO: 1). Optionally, in a non-limiting example, the linker consists essentially of SGGGGG (SEQ ID NO: 1). Optionally, in a non-limiting example, the linker consists of SGGGGG (SEQ ID NO: 1).

[0083] In some embodiments, exemplary sequences for the Fos leucine zipper dimerization motif and the Jun leucine zipper dimerization motif are set forth in SEQ ID NOs: 23 and 24, respectively.

[0084] As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can comprise a sequence 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 100% identical to the sequence set forth in SEQ ID NO: 23. As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can consist essentially of a sequence 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 100% identical to the sequence set forth in SEQ ID NO: 23. As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can consist of a sequence 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 100% identical to the sequence set forth in SEQ ID NO: 23. As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can comprise a sequence 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 set forth in SEQ ID NO: 23. As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can consist essentially of a sequence 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 set forth in SEQ ID NO:23.As a non-limiting example, the Fos leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can 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 set forth in SEQ ID NO:23.

[0085] Similarly, in some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can comprise a sequence 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 100% identical to the sequence set forth in SEQ ID NO: 24. Similarly, in some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can consist essentially of a sequence 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 100% identical to the sequence set forth in SEQ ID NO: 24. Similarly, in some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can consist of a sequence 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 100% identical to the sequence set forth in SEQ ID NO: 24. Similarly, in some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can comprise a sequence 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 set forth in SEQ ID NO: 24.Similarly, in some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can consist essentially of a sequence 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 set forth in SEQ ID NO: 24. Similarly, in some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed in some embodiments herein can consist of a sequence 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 set forth in SEQ ID NO: 24.

[0086] In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein is 90% to 100% identical to the sequence set forth in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein is 92% to 100% identical to the sequence set forth in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein is 94% to 100% identical to the sequence set forth in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein is 96% to 100% identical to the sequence set forth in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein is 98% to 100% identical to the sequence set forth in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein is 90% to 98% identical to the sequence set forth in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein is 90% to 96% identical to the sequence set forth in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein is 90% to 94% identical to the sequence set forth in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein is 90% to 92% identical to the sequence set forth in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein is 92% to 98% identical to the sequence set forth in SEQ ID NO:23. In some embodiments, the Fos leucine zipper dimerization motif used in the compositions disclosed herein is 94% to 96% identical to the sequence set forth in SEQ ID NO:23.

[0087] In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein is 90% to 100% identical to the sequence set forth in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein is 92% to 100% identical to the sequence set forth in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein is 94% to 100% identical to the sequence set forth in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein is 96% to 100% identical to the sequence set forth in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein is 98% to 100% identical to the sequence set forth in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein is 90% to 98% identical to the sequence set forth in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein is 90% to 96% identical to the sequence set forth in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein is 90% to 94% identical to the sequence set forth in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein is 90% to 92% identical to the sequence set forth in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein is 92% to 98% identical to the sequence set forth in SEQ ID NO:24. In some embodiments, the Jun leucine zipper dimerization motif used in the compositions disclosed herein is 94% to 96% identical to the sequence set forth in SEQ ID NO:24.

[0088] 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 using an immunoglobulin scaffold (e.g., an IgG scaffold). 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, are linked to an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, respectively, or vice versa. See, e.g., Hamad et al. (1998) J. Exp. Med. 188(9):1633-1640, which is incorporated herein by reference in its entirety for all purposes. In another exemplary embodiment, the hydrophobic transmembrane region of the MHC class II α chain and the hydrophobic transmembrane region of the MHC class II β chain are replaced by an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, respectively, or vice versa. In another exemplary embodiment, the extracellular domain of an MHC class II α chain or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II α1 domain or a portion or fragment or variant thereof, or an MHC class II α1 and α2 domains or portions or fragments or variants thereof) can be linked to an immunoglobulin light chain variable region (e.g., including but not limited to, fused in frame or fused via a linker), and the extracellular domain of an MHC class II β chain or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II β1 domain or a portion or fragment or variant thereof, or an MHC class II β1 and β2 domains or portions or fragments or variants thereof) can be linked to an immunoglobulin heavy chain variable region (e.g., including but not limited to, fused in frame or fused via a linker).In another exemplary embodiment, the extracellular domain of an MHC class II α chain or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II α1 domain or a portion or fragment or variant thereof, or an MHC class II α1 and α2 domains or portions or fragments or variants thereof) can be linked to an immunoglobulin heavy chain variable region (e.g., including but not limited to, fused in frame or fused via a linker), and the extracellular domain of an MHC class II β chain or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II β1 domain or a portion or fragment or variant thereof, or an MHC class II β1 and β2 domains or portions or fragments or variants thereof) can be linked to an immunoglobulin light chain variable region (e.g., including but not limited to, fused in frame or fused via a linker). The linkages (for example, but not limited to, fusion in frame or fusion via a linker) can be placed, for example, at the C-terminus of the extracellular domain of the MHC class II α chain, or a portion, fragment, or variant thereof, and at the C-terminus of the extracellular domain of the MHC class II β chain, or a portion, fragment, or variant thereof. Suitable linkers are disclosed in more detail elsewhere herein.

[0089] In some embodiments, the MHC class II α chain, or a portion, fragment, or variant thereof, and / or the MHC class II β chain, or a portion, fragment, or variant thereof, can be linked to an immunoglobulin fragment crystallizable (Fc) region or fragment (e.g., an IgG2a Fc domain, such as, but not limited to, a murine IgG2a Fc domain). See, e.g., 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 can include a Davis-body modification (e.g., a CH3 modification that enables differential binding of the Fc to Protein A) to facilitate purification. See, e.g., U.S. Patent No. 8,586,713, incorporated herein by reference in its entirety for all purposes. As a non-limiting example, the Fc segment used may comprise a hinge, C H 2, and C H 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 replace the F(ab) arm of an antibody. The hinge region may comprise, for example, a C H 2 and C HThe mobility of the MHC class II α chain, or a portion, fragment, or variant thereof, and / or the MHC class II β chain, or a portion, fragment, or variant thereof, can be increased compared to an Fc segment comprising three domains. In exemplary embodiments, 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 an MHC class II α chain or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II α1 domain or a portion or fragment or variant thereof, or an MHC class II α1 and α2 domains or portions or fragments or variants thereof) can be linked to an immunoglobulin Fc region or fragment (e.g., including but not limited to, fused in frame or fused via a linker), and / or the extracellular domain of an MHC class II β chain or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II β1 domain or a portion or fragment or variant thereof, or an MHC class II β1 and β2 domains or portions or fragments or variants thereof) can be linked to an immunoglobulin Fc region or fragment (e.g., including but not limited to, fused in frame or fused via a linker). The linkages (for example, but not limited to, fusion in frame or fusion via a linker) can be placed, for example, at the C-terminus of the extracellular domain of the MHC class II α chain, or a portion, fragment, or variant thereof, and at the C-terminus of the extracellular domain of the MHC class II β chain, or a portion, fragment, or variant thereof. Suitable linkers are disclosed in more detail elsewhere herein.

[0090] Optionally, in some embodiments, the MHC class II α chain, or a portion, fragment, or variant thereof, and / or the MHC class II β chain, or a portion, fragment, or variant thereof, can be further linked to an immunoglobulin fragment crystallizable (Fc) region, or a portion, fragment, or variant thereof (e.g., without limitation, allowing for the production of bivalent molecules). See, e.g., 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. As a non-limiting example, the Fc segment used may comprise a hinge, C H 2, and C H The immunoglobulin Fc region or fragment may comprise three domains. In one exemplary embodiment, the immunoglobulin Fc region or fragment may be linked (for example, but not limited to, via a fusion or linker) to the C-terminus of the Fos leucine zipper dimerization motif and / or to the C-terminus of the Jun leucine zipper dimerization motif. Suitable linkers are disclosed elsewhere herein.

[0091] In another exemplary embodiment, 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, are linked using a knobs-into-holes strategy. Knobs-into-holes is a heterodimerization strategy in which knobs and hole variations are designed to heterodimerize by inserting knobs into appropriately designed holes in the partner chain or domain. See, e.g., 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, knobs and holes can be linked using an immunoglobulin Fc region or fragment (e.g., C H3 domains). As another non-limiting example, a knob can be placed on an MHC class II α chain, or portion, fragment, or variant thereof, designed to insert into a corresponding hole in an MHC class II β chain, or 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 hole of the same or similar size as the knob can be created by replacing an amino acid with a large side chain with an amino acid with a small side chain. As a non-limiting example, a knob can be constructed by replacing an amino acid with a small side chain with tyrosine or tryptophan, and a corresponding hole can be constructed by replacing an amino acid with a large side chain with alanine or threonine.

[0092] 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 charge mutations. 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 or neutral amino acids. Charged amino acids are amino acid residues with charged side chains. These can be either positively charged side chains, such as those found in arginine (Arg, R), histidine (His, H), and lysine (Lys, K), or negatively charged side chains, such as those found in aspartic acid (Asp, D) and glutamic acid (Glu, E). Neutral amino acids are 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). By way of non-limiting example, one or more positively charged amino acids can be engineered into an MHC class II α chain, or portion or fragment or variant thereof, to interact with one or more negatively charged amino acids of an MHC class II β chain, or portion or 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 or fragment or variant thereof, to interact with one or more positively charged amino acids of the MHC class II β chain, or a portion or fragment or variant thereof, or vice versa.As another non-limiting example, one or more negatively charged amino acids can be engineered into an MHC class II α chain, or a portion, fragment, or variant thereof, to interact with one or more positively charged amino acids engineered into an 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 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, by substituting a neutral amino acid residue with a charged amino acid residue. In some embodiments, charged amino acids can be engineered into 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, by substituting a charged amino acid residue for an oppositely charged amino acid residue (e.g., substituting a negatively charged amino acid residue with a positively charged amino acid residue, or substituting a positively charged amino acid residue with a negatively charged amino acid residue). In one embodiment, the MHC can be linked to an immunoglobulin Fc region containing mutations with different charges. See, for example, US Pat. No. 9,358,286, which is incorporated herein by reference in its entirety for all purposes.

[0093] In another 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 covalently linked (e.g., by a linker, such as, but not limited to, a peptide linker). See, for example, Burrows et al. (1999) Protein Eng. 12(9):771-778, incorporated herein by reference in its entirety for all purposes. Such an MHC class II molecule can be a single-chain MHC fusion. In one exemplary embodiment, the single-chain MHC fusion can be a minimal TCR binding unit containing only the α1 and β1 domains, or a portion, fragment, or variant thereof (or excluding the α2 and β2 domains, and excluding the transmembrane or cytoplasmic domains of the α and β chains). In an exemplary embodiment, the hydrophobic transmembrane regions of the MHC class II α chain and the MHC class II β chain are replaced by a linker, such as a peptide linker. In one exemplary embodiment, the extracellular domain of an MHC class II α chain, or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II α1 domain, or a portion or fragment or variant thereof, or an MHC class II α1 and α2 domain, or a portion or fragment or variant thereof) can be linked (e.g., including but not limited to, fused in frame or fused via a linker) to the extracellular domain of an MHC class II β chain, or a portion or fragment or variant thereof (e.g., including but not limited to, an MHC class II β1 domain, or a portion or fragment or variant thereof, or an MHC class II β1 and β2 domain, 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 linked 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, can be linked 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.

[0094] B. MHC Ligand Peptide and Binding to MHC Class II Molecules In some embodiments of the present invention, the MHC ligand peptide in a peptide-MHC class II complex can include any peptide (i.e., antigenic peptide) that can bind to an MHC protein in such a way that the MHC-peptide complex can bind to a T cell receptor (TCR) and influence T cell responses. The characteristics of an antigenic peptide, such as its length and amino acid composition, can depend on several factors, including, but not limited to, the peptide's ability to fit into the peptide-binding groove, the experimental conditions, and the antigen of interest. These factors can be determined using commercially available computer programs such as Protean II™ (Proteus) and SPOT™. Binding of a 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 the pMHC-binding protein produced by an animal. Such spatial control is due in part to hydrogen bonds formed between the peptide and the MHC protein. Based on knowledge of how peptides bind to various MHCs, key MHC anchor amino acids and surface-exposed amino acids that vary among various peptides can be determined. Peptide binding to MHC class II molecules is stabilized by hydrophobic engagement and hydrogen bond formation. Peptides adopt a type II polyproline helix to interact with the binding groove. Without wishing to be bound by theory, this conformation is thought to cause the peptide to twist in a specific way, sequestering the peptide side chains in a polymorphic pocket of the MHC II protein. See, e.g., Ferrante and Gorski (2007) J. Immunol. 178:7181-7189, incorporated herein by reference in its entirety for all purposes. Without wishing to be bound by theory, these pockets are generally thought to accommodate the side chains of peptide residues at positions P1, P4, P6, and P9, and have been identified as major anchors. In addition to these interactions, small pockets or shelves in the binding site accommodating residues P2, P3, P7, and P10 are recognized as minor or auxiliary anchors.

[0095] In some embodiments, non-limiting examples of MHC ligand peptides suitable for use in the disclosed peptide-MHC class II complexes include peptides comprising residues P-3 through P12. In some embodiments, non-limiting examples of MHC ligand peptides suitable for use in the disclosed peptide-MHC class II complexes include peptides consisting essentially of residues P-3 through P12. In some embodiments, non-limiting examples of MHC ligand peptides suitable for use in the disclosed peptide-MHC class II complexes include peptides consisting of residues P-3 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-2 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-2 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-2 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P1 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P1 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P1 through P9.

[0096] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-3 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-2 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-3 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-2 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-3 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-2 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-3 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-2 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P9.

[0097] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-3 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-2 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-3 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-2 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-2 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-3 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-2 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P9.

[0098] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-3 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-2 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-3 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-2 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-2 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-3 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-2 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P1 through P9.

[0099] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-3 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-3 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-3 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-3 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-2 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-2 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-2 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-2 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P-1 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides comprising residues P1 through P9.

[0100] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-3 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-3 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-3 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-3 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-2 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-2 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-2 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-2 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P-1 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting essentially of residues P1 through P9.

[0101] In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-3 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-3 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-3 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-3 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-2 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-2 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-2 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-2 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P-1 through P9. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P1 through P12. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P1 through P11. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P1 through P10. In some embodiments, non-limiting examples of suitable MHC ligand peptides include peptides consisting of residues P1 through P9.

[0102] In one embodiment, the MHC ligand peptide comprises residues P1 through P12. In one embodiment, the MHC ligand peptide consists essentially of residues P1 through P12. In one embodiment, the MHC ligand peptide consists of residues P1 through P12. In one embodiment, the MHC ligand peptide comprises residues P-1 through P11. In one embodiment, the MHC ligand peptide consists essentially of residues P-1 through P11. In one embodiment, the MHC ligand peptide consists of residues P-1 through P11. In one embodiment, the MHC ligand peptide comprises residues P-1 through P9. In one embodiment, the MHC ligand peptide consists essentially of residues P-1 through P9. In one embodiment, the MHC ligand peptide comprises residues P-3 through P9. In one embodiment, the MHC ligand peptide consists essentially of residues P-3 through P9. In one embodiment, the MHC ligand peptide consists essentially of residues P-3 through P9. In one embodiment, the MHC ligand peptide consists of residues P-3 through P9.

[0103] In some embodiments, non-limiting examples of antigenic peptides suitable for use in the disclosed peptide-MHC class II complexes include peptides comprising an antigen, or a portion, fragment, or variant thereof, selected from the group consisting of an autoantigen, a tumor-associated antigen, an infectious agent, a toxin, an allergen, or a combination thereof. In one exemplary embodiment, the MHC ligand peptide comprises at least a portion, fragment, or variant (e.g., without limitation, an antigenic determinant) of a human self-protein associated with an autoimmune disorder. In another embodiment, the MHC ligand peptide comprises at least a portion, fragment, or variant (e.g., without limitation, an antigenic determinant) of a protein of an infectious pathogen (e.g., without limitation, a bacterium, a virus, or a parasite). In another embodiment, the MHC ligand peptide comprises at least a portion, fragment, or variant (e.g., without limitation, an antigenic determinant) of an allergen. In another embodiment, the MHC ligand peptide comprises at least a portion, fragment, or variant (e.g., without limitation, an antigenic determinant) of a tumor-associated protein. In another embodiment, the MHC ligand peptide is associated with a T cell-mediated disease (e.g., a T cell-mediated autoimmune disease such as, but not limited to, type 1 diabetes mellitus, rheumatoid arthritis, multiple sclerosis, celiac disease, Addison's disease, and hypothyroidism). In one embodiment, the MHC ligand peptide can 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) can 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., a gliadin peptide or a gliadin-derived peptide) can consist essentially 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., a gliadin peptide or gliadin-derived peptide) can 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., a gliadin peptide or gliadin-derived peptide) can include 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., a gliadin peptide or gliadin-derived peptide) can 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., a gliadin peptide or gliadin-derived peptide) can consist 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).

[0104] In some embodiments, the MHC ligand peptide can be of any suitable length to bind to an MHC protein in a manner that allows the MHC-peptide complex to bind to the TCR and result in 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 in integer increments of 5 to 40 amino acids in length (i.e., 5, 6, 7, 8, 9, ... 40)). Traditionally, MHC class II binding peptides vary from about 9 to about 40 amino acids, but in almost all cases, the peptides can be truncated to a core of 9 to 11 amino acids without loss of MHC binding activity or T cell recognition. In some embodiments, the length of the MHC ligand peptide can be from about 5 to about 40 amino acids. In some embodiments, the length of the MHC ligand peptide can be from about 10 to about 40 amino acids. In some embodiments, the MHC ligand peptide can be about 15 to about 40 amino acids in length. In some embodiments, the MHC ligand peptide can be about 20 to about 40 amino acids in length. In some embodiments, the MHC ligand peptide can be about 25 to about 40 amino acids in length. In some embodiments, the MHC ligand peptide can be about 30 to about 40 amino acids in length. In some embodiments, the MHC ligand peptide can be about 35 to about 40 amino acids in length. In some embodiments, the MHC ligand peptide can be about 5 to about 35 amino acids in length. In some embodiments, the MHC ligand peptide can be about 5 to about 30 amino acids in length. In some embodiments, the MHC ligand peptide can be about 5 to about 25 amino acids in length. In some embodiments, the MHC ligand peptide can be about 5 to about 20 amino acids in length. In some embodiments, the MHC ligand peptide can be about 5 to about 15 amino acids in length.In some embodiments, the MHC ligand peptide can be about 5 to about 10 amino acids in length. In some embodiments, the MHC ligand peptide can be about 10 to about 35 amino acids in length. In some embodiments, the MHC ligand peptide can be about 15 to about 30 amino acids in length. In some embodiments, the MHC ligand peptide can be about 20 to about 25 amino acids in length. In some embodiments, the MHC ligand peptide can be about 9 to about 11 amino acids in length. In some embodiments, the MHC ligand peptide can be about 10 to about 18 amino acids in length. In some embodiments, the MHC ligand peptide can be about 9 to about 15 amino acids in length. In some embodiments, the MHC ligand peptide can be about 9 to about 14 amino acids in length. In some embodiments, the MHC ligand peptide can be about 9 to about 13 amino acids in length. In some embodiments, the MHC ligand peptide can be about 9 to about 12 amino acids in length. In some embodiments, the MHC ligand peptide can be about 10 to about 15 amino acids in length. In some embodiments, the MHC ligand peptide can be about 10 to about 14 amino acids in length. 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.

[0105] In some embodiments, the MHC ligand peptide can be of any suitable length to bind to an MHC protein in a manner that allows the MHC-peptide complex to bind to the TCR and result in 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 in integer increments of 5 to 40 amino acids in length (i.e., 5, 6, 7, 8, 9, ... 40)). Traditionally, MHC class II binding peptides vary from 9 to 40 amino acids, but in almost all cases, peptides can be truncated to the core 9 to 11 amino acids without loss of MHC binding activity or T cell recognition. In some embodiments, the length of the MHC ligand peptide can be 5 to 40 amino acids. In some embodiments, the length of the MHC ligand peptide can be 10 to 40 amino acids. In some embodiments, the length of the MHC ligand peptide can be 15 to 40 amino acids. In some embodiments, the MHC ligand peptide can be 20-40 amino acids in length. In some embodiments, the MHC ligand peptide can be 25-40 amino acids in length. In some embodiments, the MHC ligand peptide can be 30-40 amino acids in length. In some embodiments, the MHC ligand peptide can be 35-40 amino acids in length. In some embodiments, the MHC ligand peptide can be 5-35 amino acids in length. In some embodiments, the MHC ligand peptide can be 5-30 amino acids in length. In some embodiments, the MHC ligand peptide can be 5-25 amino acids in length. In some embodiments, the MHC ligand peptide can be 5-20 amino acids in length. In some embodiments, the MHC ligand peptide can be 5-15 amino acids in length. In some embodiments, the MHC ligand peptide can be 5-10 amino acids in length.In some embodiments, the MHC ligand peptide can be 10-35 amino acids in length. In some embodiments, the MHC ligand peptide can be 15-30 amino acids in length. In some embodiments, the MHC ligand peptide can be 20-25 amino acids in length. In some embodiments, the MHC ligand peptide can be 9-11 amino acids in length. In some embodiments, the MHC ligand peptide can be 10-18 amino acids in length. In some embodiments, the MHC ligand peptide can be 9-15 amino acids in length. In some embodiments, the MHC ligand peptide can be 9-14 amino acids in length. In some embodiments, the MHC ligand peptide can be 9-13 amino acids in length. In some embodiments, the MHC ligand peptide can be 9-12 amino acids in length. In some embodiments, the MHC ligand peptide can be 10-15 amino acids in length. In some embodiments, the MHC ligand peptide can be 10-14 amino acids in length. In some embodiments, the MHC ligand peptide can be 10-13 amino acids in length. In some embodiments, the MHC ligand peptide can be 10-12 amino acids in length.

[0106] (1) Linker 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 as a fusion protein. In exemplary embodiments, the MHC class II molecule (e.g., 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 connected via a linker (e.g., covalently linked, such as, but not limited to, a peptide linker). As non-limiting examples, the MHC ligand peptide can be directly or indirectly connected to the N-terminus of the MHC class II β chain, or a portion, fragment, or variant thereof, the C-terminus of the MHC class II β chain, or a portion, fragment, or variant thereof, the N-terminus of the MHC class II α chain, or a portion, fragment, or variant thereof, or the C-terminus of the MHC class II α chain, or a portion, fragment, or variant thereof. In exemplary embodiments, the MHC ligand peptide can be directly or indirectly connected to the N-terminus of the MHC class II β chain, or a portion, fragment, or variant thereof. As a non-limiting example, a peptide-MHC class II complex can include, from amino to carboxy terminus, an MHC ligand peptide, a linker, and an MHC class II β chain or a portion, fragment, or variant thereof. As a non-limiting example, the linker can extend from the C-terminus of the MHC ligand peptide to the N-terminus of the MHC class II β chain or a portion, fragment, or variant thereof. The linker can be constructed so that the bound MHC ligand peptide folds into the binding groove of the MHC class II molecule, resulting in a functional peptide-MHC class II complex. Linking a peptide to an MHC class II molecule via a flexible linker offers the advantage of ensuring that the peptide occupies and remains bound to the MHC during biosynthesis, transport, and presentation.

[0107] The length of the linker connecting the MHC ligand peptide to the MHC class II molecule can be any suitable length. In exemplary embodiments, the linker can be long enough to allow the MHC ligand peptide to reach and bind to the peptide-binding groove of the MHC class II molecule, yet short enough so that the linker does not substantially inhibit binding between the MHC ligand peptide and the peptide-binding groove of the MHC class II molecule. The length of the linker can be designed to extend beyond the distance between the N- and C-termini to be bound based on known structural information (e.g., but not limited to, known tertiary structural information). The appropriate size and sequence of the linker can also be determined by conventional computer modeling techniques based on the predicted tertiary structure of the peptide-MHC class II complex. As a non-limiting example, from the HLA-DQ structure deposited under 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 either the MHC α subunit or the MHC β subunit is approximately 30 Å. See, for example, Kim et al. (2004) Proc. Natl. Acad. Sci. USA 101(12):4175-4179, which is incorporated herein by reference in its entirety for all purposes. Thus, when the C-terminus of an MHC ligand peptide is connected to the N-terminus of an MHC class II β chain, or a portion, domain, fragment, or variant thereof, or an MHC class II α chain, or a portion, domain, fragment, or variant thereof, via a linker, 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, domain, fragment, or variant thereof, when the MHC ligand peptide is positioned within the peptide-binding groove of the MHC class II molecule. From the above measurements, for example, a linker of more than 30 Å (3.5 Å per amino acid residue = 9 amino acids) may be required to extend the measured distance. Additional amino acids can also be included to allow the linker to avoid the protein molecule surface between the two connection points.

[0108] As non-limiting examples, a 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 in length. Similarly, in some embodiments, a linker can be about 50 amino acids or less, about 45 amino acids or less, about 40 amino acids or less, about 35 amino acids or less, about 30 amino acids or less, about 25 amino acids or less, about 20 amino acids or less, or about 15 amino acids or less in length. In some embodiments, a linker can be about 9 amino acids to about 50 amino acids in length. In some embodiments, a linker can be about 9 amino acids to about 45 amino acids in length. In some embodiments, a linker can be about 9 amino acids to about 40 amino acids in length. In some embodiments, a linker can be about 9 amino acids to about 35 amino acids in length. In some embodiments, a linker can be about 9 amino acids to about 30 amino acids in length. In some embodiments, a linker can be about 9 amino acids to about 25 amino acids in length. In some embodiments, the linker can be about 9 amino acids to about 20 amino acids in length. In some embodiments, the linker can be about 10 amino acids to about 45 amino acids in length. In some embodiments, the linker can be about 10 amino acids to about 40 amino acids in length. In some embodiments, the linker can be about 10 amino acids to about 35 amino acids in length. In some embodiments, the linker can be about 10 amino acids to about 30 amino acids in length. In some embodiments, the linker can be about 10 amino acids to about 25 amino acids in length. In some embodiments, the linker can be about 10 amino acids to about 20 amino acids in length. In some embodiments, the linker can be about 15 amino acids to about 45 amino acids in length. In some embodiments, the linker can be about 15 amino acids to about 40 amino acids in length. In some embodiments, the linker can be about 15 amino acids to about 35 amino acids in length.In some embodiments, the linker can be about 15 to about 30 amino acids in length. In some embodiments, the linker can be about 15 to about 25 amino acids in length. In some embodiments, the linker can be about 15 to about 20 amino acids in length. In some embodiments, the linker can be about 10 to about 50 amino acids in length. In some embodiments, the linker can be about 15 to about 50 amino acids in length. In some embodiments, the linker can be about 20 to about 50 amino acids in length. In some embodiments, the linker can be about 25 to about 50 amino acids in length. In some embodiments, the linker can be about 30 to about 50 amino acids in length. In some embodiments, the linker can be about 35 to about 50 amino acids in length. In some embodiments, the linker can be about 40 to about 50 amino acids in length. In some embodiments, the linker can be about 45 to about 50 amino acids in length. In some embodiments, the linker can be about 10 to about 20 amino acids in length. In some embodiments, the linker can be about 11 amino acids to about 19 amino acids in length. In some embodiments, the linker can be about 12 amino acids to about 18 amino acids in length. In some embodiments, the linker can be about 13 amino acids to about 17 amino acids in length. In some embodiments, the linker can be about 14 amino acids to about 16 amino acids in length. In some embodiments, the linker can vary in size from 9 to 50 amino acids in whole-number increments (i.e., 9, 10, 11, 12, 13, ... 50) peptide length. In an exemplary embodiment, the linker can be about 15 amino acids in length.

[0109] As non-limiting examples, a 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 long. Similarly, in some embodiments, a linker can be 50 amino acids or less, 45 amino acids or less, 40 amino acids or less, 35 amino acids or less, 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, or 15 amino acids or less in length. In some embodiments, a linker can be 9 to 50 amino acids in length. In some embodiments, a linker can be 9 to 45 amino acids in length. In some embodiments, a linker can be 9 to 40 amino acids in length. In some embodiments, a linker can be 9 to 35 amino acids in length. In some embodiments, a linker can be 9 to 30 amino acids in length. In some embodiments, a linker can be 9 to 25 amino acids in length. In some embodiments, a linker can be 9 to 20 amino acids in length. In some embodiments, a linker can be 10 to 45 amino acids in length. In some embodiments, the linker can be 10 to 40 amino acids in length. In some embodiments, the linker can be 10 to 35 amino acids in length. In some embodiments, the linker can be 10 to 30 amino acids in length. In some embodiments, the linker can be 10 to 25 amino acids in length. In some embodiments, the linker can be 10 to 20 amino acids in length. In some embodiments, the linker can be 15 to 45 amino acids in length. In some embodiments, the linker can be 15 to 40 amino acids in length. In some embodiments, the linker can be 15 to 35 amino acids in length. In some embodiments, the linker can be 15 to 30 amino acids in length.In some embodiments, the linker can be 15 to 25 amino acids in length. In some embodiments, the linker can be 15 to 20 amino acids in length. In some embodiments, the linker can be 10 to 50 amino acids in length. In some embodiments, the linker can be 15 to 50 amino acids in length. In some embodiments, the linker can be 20 to 50 amino acids in length. In some embodiments, the linker can be 25 to 50 amino acids in length. In some embodiments, the linker can be 30 to 50 amino acids in length. In some embodiments, the linker can be 35 to 50 amino acids in length. In some embodiments, the linker can be 40 to 50 amino acids in length. In some embodiments, the linker can be 45 to 50 amino acids in length. In some embodiments, the linker can be 10 to 20 amino acids in length. In some embodiments, the linker can be 11 to 19 amino acids in length. In some embodiments, the linker can be 12 to 18 amino acids in length. In some embodiments, the linker can be 13 to 17 amino acids in length. In some embodiments, the linker can be 14 to 16 amino acids in length. In some embodiments, the linker can vary in size from 9 to 50 amino acids in length in integer increments (i.e., 9, 10, 11, 12, 13, ... 50). In an exemplary embodiment, the linker can be 15 amino acids in length.

[0110] Any suitable amino acid can be used in the linker. In some embodiments, for non-limiting examples of suitable linkers, including flexible linkers, rigid linkers, and cleavable linkers, see, for example, Chen et al. (2013) Adv. Drug Deliv. Rev. 65(10):1357-1369. The PDB database can also be searched for amino acid sequences that can span a defined distance between protein segments or domains, as shown on the server of the Center for Integrative Bioinformatics at the University of Amsterdam at ibi.vu.nl / programs / linkerdbwww.

[0111] The linkers used in the peptide-MHC class II complexes disclosed in some embodiments herein may have one or more or all of the following characteristics: the linker is flexible, the linker is non-immunogenic, the linker does not contain charged amino acids, the linker contains polar amino acids, and any combination thereof. Flexibility allows MHC ligand peptides to freely bind and assemble into the natural peptide-binding groove of MHC class II molecules. Flexibility can be achieved, for example, by using linkers 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 can 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 can be glycine. In some embodiments, about 40% to about 80% of the amino acids in the linker can be glycine. In some embodiments, 40% to 80% of the amino acids in the linker can be glycine. In some embodiments, about 50% to about 80% of the amino acids in the linker can be glycine. In some embodiments, 50% to 80% of the amino acids in the linker can be glycine. In some embodiments, about 60% to about 80% of the amino acids in the linker can be glycine. In some embodiments, 60% to 80% of the amino acids in the linker can be glycine. In some embodiments, about 70% to about 80% of the amino acids in the linker can be glycine. In some embodiments, 70% to 80% of the amino acids in the linker can be glycine. In some embodiments, about 40% to about 70% of the amino acids in the linker can be glycine.In some embodiments, 40% to 70% of the amino acids in the linker can be glycine. In some embodiments, about 40% to about 60% of the amino acids in the linker can be glycine. In some embodiments, 40% to 60% of the amino acids in the linker can be glycine. In some embodiments, about 40% to about 50% of the amino acids in the linker can be glycine. In some embodiments, 40% to 50% of the amino acids in the linker can be glycine. In some embodiments, about 50% to about 70% of the amino acids in the linker can be glycine. In some embodiments, 50% to 70% of the amino acids in the linker can be glycine. In some embodiments, about 55% to about 65% of the amino acids in the linker can be glycine. Inclusion of a polar amino acid can 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 exemplary embodiments, one or more serines are included in the linker. Omitting charged amino acids (e.g., Lys, Arg, Glu, and Asp) helps 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., including, but not limited to, flexible amino acids such as GIy) 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 comprises 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 include 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 include 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 include 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, and does not include 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, and does not include any charged amino acids.

[0112] In some embodiments, a linker suitable for use in the disclosed peptide-MHC class II complexes is a cleavable linker that includes a cleavage site. As a non-limiting example, the linker can include the tobacco etch virus (TEV) protease cleavage site ENLYFQ (SEQ ID NO: 22). However, other cleavage sites (e.g., thrombin-sensitive cleavage sites, furin-sensitive cleavage sites, rhinovirus 3C protease cleavage sites, or enteropeptidase cleavage sites) can also be used. For example, see Waugh (2011) Protein Immunoglobulin Genetics, vol. 1, pp. 111-114, which is incorporated herein by reference in its entirety for all purposes. See Expr. Purif. 80(2):283-293.

[0113] Some linkers suitable for use in 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 (e.g., (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 one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. (GGGGS) n The (SEQ ID NO: 4) linker is particularly suitable because it contains both a flexible amino acid (Gly) and a polar amino acid that can form hydrogen bonds (Ser) to improve solubility. A suitable linker is (GSGGS) n (SEQ ID NO: 2), (GGGS) n (SEQ ID NO: 3), and (GGGGS) n (SEQ ID NO: 4). A suitable linker may essentially be (GSGGS) n (SEQ ID NO: 2), (GGGS) n (SEQ ID NO: 3), and (GGGGS) n (SEQ ID NO: 4). A suitable linker may consist of either (GSGGS) n (SEQ ID NO: 2), (GGGS) n (SEQ ID NO: 3), and (GGGGS) n(SEQ ID NO: 4). In some embodiments, the peptide linker (e.g., a peptide linker linking an MHC ligand peptide to an MHC class II molecule) can comprise about two to about four repeats of the sequence set forth in SEQ ID NO: 4. In some embodiments, the peptide linker (e.g., a peptide linker linking an MHC ligand peptide to an MHC class II molecule) can comprise about two to about four repeats of the sequence set forth in SEQ ID NO: 4, wherein one amino acid in one of the repeats is mutated to cysteine. In some embodiments, the peptide linker (e.g., a peptide linker linking an MHC ligand peptide to an MHC class II molecule) can comprise about two to four repeats of the sequence set forth in SEQ ID NO: 4, wherein one amino acid in one of the repeats is mutated to cysteine. As a non-limiting example, cysteine ​​can be the first, second, third, or fourth amino acid of a linker (e.g., but not limited to, the second amino acid of a linker). Glycine and glycine-serine polymers can be used. Both glycine and serine are relatively unstructured and therefore function as neutral tethers between components. Glycine has access to much more phi-psi space than alanine, making it much less restrictive than residues with longer side chains.In some embodiments, exemplary linkers can include amino acid sequences such as, but not limited to, GGSG (SEQ ID NO:5), GGSGG (SEQ ID NO:6), GSGSG (SEQ ID NO:7), GSGGG (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), GGGGSGGGGSGGGGGS (SEQ ID NO:16), or GGGASGGGGS (SEQ ID NO:17), GGGGSGGGGSGGGGS (SEQ ID NO:18), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:19), GCGGS (SEQ ID NO:20), GCGGSGGGGSGGGGS (SEQ ID NO:21), GGGGSENLYFQGGGGS (SEQ ID NO:47). In some embodiments, exemplary linkers can consist essentially of an amino acid sequence such as, but not limited to, GGSG (SEQ ID NO:5), GGSGG (SEQ ID NO:6), GSGSG (SEQ ID NO:7), GSGGG (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), GGGGSGGGGSGGGGGS (SEQ ID NO:16), or GGGASGGGGS (SEQ ID NO:17), GGGGSGGGGSGGGGS (SEQ ID NO:18), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:19), GCGGS (SEQ ID NO:20), GCGGSGGGGSGGGGS (SEQ ID NO:21), GGGGSENLYFQGGGGS (SEQ ID NO:47).In some embodiments, exemplary linkers can consist of amino acid sequences such as, but not limited to, GGSG (SEQ ID NO:5), GGSGG (SEQ ID NO:6), GSGSG (SEQ ID NO:7), GSGGG (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), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:19), GCGGS (SEQ ID NO:20), GCGGSGGGGSGGGGS (SEQ ID NO:21), GGGGSENLYFQGGGGS (SEQ ID NO:47). In an exemplary embodiment, the linker (e.g., the linker linking the MHC ligand peptide to the MHC class II molecule) comprises GCGGSGGGGSGGGGS (SEQ ID NO: 21). In an exemplary embodiment, the linker (e.g., the linker linking the MHC ligand peptide to the MHC class II molecule) consists essentially of GCGGSGGGGSGGGGS (SEQ ID NO: 21). In an exemplary embodiment, the linker (e.g., the linker linking the MHC ligand peptide to the MHC class II molecule) consists of GCGGSGGGGSGGGGS (SEQ ID NO: 21).

[0114] In some embodiments of the linker, the linker polypeptide comprises a cysteine ​​residue capable of forming a disulfide bond with a cysteine ​​residue present in an MHC class II molecule. In exemplary embodiments, the linker can comprise a cysteine ​​residue capable of forming a disulfide bond with a cysteine ​​residue present in 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. As a non-limiting example, the cysteine ​​can be the first, second, third, or fourth amino acid of the linker (e.g., without limitation, the second amino acid of the linker).

[0115] Although the above linkers are described for linking an MHC class II molecule (e.g., an MHC class II β chain or a portion or fragment or variant thereof, or an MHC class II α chain or a portion or fragment or variant thereof) to an MHC ligand peptide, these linkers can also be used in any other context described herein in which a linker is used.

[0116] (2) Disulfide bridge In some embodiments of the complex, the MHC ligand peptide or the linker connecting the MHC ligand peptide to the MHC class II molecule is linked to at least one chain of the MHC class II molecule, or a portion, fragment, or variant thereof, via a disulfide bridge (i.e., a disulfide bond extending between a pair of oxidized cysteines). In exemplary embodiments, the MHC ligand peptide can include a first cysteine, or the linker can include a first cysteine, and the MHC class II molecule can include a second cysteine ​​proximal to the first cysteine ​​in the tertiary structure of the complex, thereby forming a disulfide bridge and linking the MHC ligand peptide 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. Proximity can be determined, for example, based on available crystal structures. Such disulfide bonds aid in positioning the MHC ligand peptide in the peptide-binding groove of the MHC class II molecule. Optionally, in some embodiments, the MHC ligand peptide can include the first cysteine ​​and no other cysteines, or the linker can include the first cysteine ​​and no other cysteines. Optionally, in some embodiments, when the MHC ligand peptide includes the first cysteine, the linker does not include other cysteines. Optionally, in some embodiments, when the linker includes the first cysteine, the MHC ligand peptide does not include other cysteines. Optionally, in some embodiments, the MHC class II molecule includes only the second cysteine ​​and does not include other cysteines or other unpaired cysteines (e.g., there are no Cys that can form disulfide bonds within the MHC class II molecule). Optionally, in some embodiments, when the second cysteine ​​is in an MHC class II α chain or portion or fragment or variant thereof, the MHC class II α chain or portion or fragment or variant thereof does not contain any other cysteines or other unpaired cysteines (e.g., no Cys that can form disulfide bonds within the MHC class II molecule).Optionally, in some embodiments, when the second cysteine ​​is in an MHC class II β chain, or portion, fragment, or variant thereof, the MHC class II β chain, or portion, fragment, or variant thereof, does not contain other cysteines or other unpaired cysteines (e.g., no Cys that can form disulfide bonds within the MHC class II molecule). The cysteine ​​in the MHC class II molecule can be a naturally occurring cysteine ​​(i.e., present in an unmodified (i.e., wild-type) MHC class II molecule) or can be a mutation relative to an unmodified (i.e., wild-type) MHC class II molecule. Similarly, in some embodiments, the cysteine ​​in the MHC ligand peptide can be a naturally occurring cysteine ​​or can be a mutation (addition or substitution) in the MHC ligand peptide. If it is a mutation in the MHC ligand peptide, it preferably faces away from the epitope formed by the peptide-MHC class II complex. The chain of the MHC class II molecule (or a portion, fragment, or variant thereof) and the chain of the MHC class II molecule (or a portion, fragment, or variant thereof) to which the linker is connected (i.e., covalently linked) and which form the disulfide bridge can be the same or different chains of the MHC class II molecule. In exemplary embodiments, 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 the MHC ligand peptide or 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 exemplary 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 the MHC ligand peptide or 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).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 or fragment or variant thereof), and a disulfide bridge can be formed between the MHC ligand peptide or linker and the α chain of the MHC class II molecule (i.e., the MHC class II α chain or a portion or fragment or variant thereof). In some embodiments, 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 or fragment or variant thereof), and a disulfide bridge can be formed between the MHC ligand peptide or linker and the β chain of the MHC class II molecule (i.e., the MHC class II β chain or a portion or fragment or variant thereof).

[0117] In some embodiments of peptide-MHC class II complexes, a non-cysteine ​​residue in the unmodified (i.e., wild-type) α chain of the MHC class II molecule (i.e., MHC class II α chain, or portion, fragment, or variant thereof) or in the unmodified (i.e., wild-type) β chain of the MHC class II molecule (i.e., MHC class II β chain, or portion, fragment, or variant thereof) can be mutated to cysteine ​​based on its proximity to a cysteine ​​in the MHC ligand peptide or in the linker connecting the MHC ligand peptide to the MHC class II molecule in the tertiary structure of the complex. In some embodiments of peptide-MHC class II complexes, a cysteine ​​residue can be inserted into the MHC class II α chain, or portion, fragment, or variant thereof, or the MHC class II β chain, or portion, fragment, or variant thereof, based on its proximity to a cysteine ​​in the MHC ligand peptide or in the linker connecting the MHC ligand peptide to the MHC class II molecule in the tertiary structure of the complex. That is, 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 (i.e., in the MHC ligand peptide or linker) that is proximal to the first cysteine ​​in the tertiary structure of the complex. Proximity can be determined, for example, based on available crystal structures. In an exemplary embodiment, position 101 of the MHC class II α chain sequence set forth in SEQ ID NO:49 or SEQ ID NO:55 or 56 can be mutated to a cysteine ​​(R101C). Non-limiting examples of MHC class II α chain sequences mutated to a cysteine ​​at this position are set forth in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:57.In some embodiments, the position in the sequence of a subject MHC class II α chain, or portion or fragment or variant thereof, corresponding to position 101 of the MHC class II α chain sequence set forth in SEQ ID NO:49 when the sequence of the subject MHC class II α chain, or portion or fragment or variant thereof, is optimally aligned with SEQ ID NO:49 can be mutated to cysteine ​​(e.g., the position corresponding to the position designated DQA1 R101 in the alignment of the full-length sequences of HLA-DPA1, HLA-DQA1, and HLA-DRA1 in Figure 3 can be mutated to cysteine). For example, position 107 of SEQ ID NO:51, or the position in the sequence of a subject MHC class II α chain, or portion or fragment or variant thereof, corresponding to position 107 of SEQ ID NO:51 when the sequence of the subject MHC class II α chain, or portion or fragment or variant thereof, is optimally aligned with SEQ ID NO:51 can be mutated to cysteine. As another example, position 101 of SEQ ID NO: 52, or the position of a subject MHC class II α chain, or portion, fragment, or variant thereof, corresponding to position 101 of SEQ ID NO: 52 when the sequence of the subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 52, can be mutated to cysteine. As yet another example, position 78 of SEQ ID NO: 59, or the position of a subject MHC class II α chain, or portion, fragment, or variant thereof, corresponding to position 78 of SEQ ID NO: 59 when the sequence of the subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 59, can be mutated to cysteine. As yet another example, position 79 of SEQ ID NO: 61, or the position of a subject MHC class II α chain, or portion, fragment, or variant thereof, corresponding to position 79 of SEQ ID NO: 61 when the sequence of the subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 61, can be mutated to cysteine.As yet another example, position 76 of SEQ ID NO: 62, or the position in the sequence of a subject MHC class II α chain, or portion, fragment, or variant thereof, corresponding to position 76 of SEQ ID NO: 62 when the sequence of the subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 62, can be mutated to cysteine. In another embodiment, position 79 of the MHC class II α chain sequence set forth in SEQ ID NO: 52 (HLA class II histocompatibility antigen, DRα chain; NCBI Accession No. P01903.1) can be mutated to cysteine ​​(F79C). A non-limiting example of an MHC class II α chain sequence with this position mutated to cysteine ​​is set forth in SEQ ID NO: 58. In some embodiments, the position in the sequence of a subject MHC class II α chain, or portion or fragment or variant thereof, that corresponds to position 79 of the MHC class II α chain sequence set forth in SEQ ID NO: 52 when the sequence of the subject MHC class II α chain, or portion or fragment or variant thereof, is optimally aligned with SEQ ID NO: 52 can be mutated to cysteine ​​(e.g., the position corresponding to the position designated DRA1 F79 in the alignment of the full-length sequences of HLA-DPA1, HLA-DQA1, and HLA-DRA1 in Figure 3 can be mutated to cysteine). For example, position 79 of SEQ ID NO: 49, or the position in the sequence of a subject MHC class II α chain, or portion or fragment or variant thereof, that corresponds to position 79 of SEQ ID NO: 49 when the sequence of the subject MHC class II α chain, or portion or fragment or variant thereof, is optimally aligned with SEQ ID NO: 49 can be mutated to cysteine. As another example, position 85 of SEQ ID NO: 51, or the position of the MHC class II α chain or portion or fragment or variant thereof of interest that corresponds to position 85 of SEQ ID NO: 51 when the sequence of the MHC class II α chain or portion or fragment or variant thereof of interest is optimally aligned with SEQ ID NO: 51, can be mutated to cysteine.As yet another example, position 56 of SEQ ID NO: 59, or the position in the sequence of a subject MHC class II α chain, or portion, fragment, or variant thereof, that corresponds to position 56 of SEQ ID NO: 59 when the sequence of the subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 59, can be mutated to cysteine. As yet another example, position 57 of SEQ ID NO: 61, or the position in the sequence of a subject MHC class II α chain, or portion, fragment, or variant thereof, that corresponds to position 57 of SEQ ID NO: 61 when the sequence of the subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 61, can be mutated to cysteine. As yet another example, position 54 of SEQ ID NO: 62, or the position in the sequence of a subject MHC class II α chain, or portion, fragment, or variant thereof, that corresponds to position 54 of SEQ ID NO: 62 when the sequence of the subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 62, can be mutated to cysteine.

[0118] In some embodiments of peptide-MHC class II complexes, cysteine ​​residues in the unmodified (i.e., wild-type) α chain of the MHC class II molecule (i.e., the MHC class II α chain, or a portion, fragment, or variant thereof) or the unmodified (i.e., wild-type) β chain of the MHC class II molecule (i.e., the MHC class II β chain, or a portion, fragment, or variant thereof) can be mutated to non-cysteine ​​residues to minimize disulfide scrambling (i.e., disulfide bonds formed between cysteine ​​residues other than those intended for use). The amino acid used in place of cysteine ​​can be selected to enable proper folding of the MHC complex. This can be determined, for example, based on available crystal structures or by evaluating 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 sterically disruptive. In an exemplary embodiment, the cysteine ​​at position 70 of the MHC class II α chain sequence set forth 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 (substituting the unpaired Cys of DQA1*0501 with Trp, Arg, or Gln of the closest MHC sequence from another species based on sequence alignment). In an exemplary embodiment, the cysteine ​​at position 70 of the MHC class II α chain sequence set forth in SEQ ID NO: 49 or 53 can be mutated to alanine (C70A). Non-limiting examples of MHC class II α chain sequences with this position mutated to alanine are set forth in SEQ ID NOs: 56 and 54. In an exemplary embodiment, the cysteine ​​at position 70 of the MHC class II α chain sequence set forth in SEQ ID NO: 49 can be mutated to glutamine (C70Q). Non-limiting examples of MHC class II α chain sequences in which this position has been mutated to glutamine are set forth in SEQ ID NOs:55 and 57.In some embodiments, a cysteine ​​in a subject MHC class II α chain sequence, or portion, fragment, or variant thereof, corresponding to position 70 in the MHC class II α chain sequence set forth in SEQ ID NO: 49 can be mutated (e.g., without limitation, to alanine or glutamine) when the subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 49 (e.g., a position corresponding to the position labeled DQA1 C70 in the alignment of the full-length sequences of HLA-DPA1, HLA-DQA1, and HLA-DRA1 in Figure 3). For example, a position in a subject MHC class II α chain, or portion, fragment, or variant thereof, corresponding to position 75 in SEQ ID NO: 51 can be mutated when the subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 51. As another example, when the sequence of a subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 52, the position of the subject MHC class II α chain, or portion, fragment, or variant thereof, corresponding to position 69 of SEQ ID NO: 52 can be mutated. As yet another example, when the sequence of a subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 59, the cysteine ​​at position 47 of the MHC class II α chain sequence set forth in SEQ ID NO: 59, or the position of the subject MHC class II α chain, or portion, fragment, or variant thereof, corresponding to position 47 of SEQ ID NO: 59 can be mutated. As yet another example, when the sequence of a subject MHC class II α chain, or portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 61, the position of the subject MHC class II α chain, or portion, fragment, or variant thereof, corresponding to position 47 of SEQ ID NO: 61 can be mutated.As yet another example, the position of the MHC class II α chain or portion or fragment or variant thereof of interest that corresponds to position 44 of SEQ ID NO: 62 when the sequence of the MHC class II α chain or portion or fragment or variant thereof of interest is optimally aligned with SEQ ID NO: 62 can be mutated.

[0119] In one exemplary embodiment, the linker connecting the MHC ligand peptide to the MHC class II molecule can include a first cysteine, and the MHC class II molecule can include a second cysteine ​​at a proximal position such that a disulfide bridge forms to link the MHC ligand peptide 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 set forth in SEQ ID NO:49 can be mutated to a cysteine ​​(R101C), or the position in the sequence of the MHC class II α chain, or portion, fragment, or variant thereof, of interest that corresponds to position 101 of the MHC class II α chain sequence set forth in SEQ ID NO:49 when the sequence of the MHC class II α chain, or portion, fragment, or variant thereof, of interest, is optimally aligned with SEQ ID NO:49 can be mutated to a cysteine. Optionally, in some embodiments, the cysteine ​​at position 70 of the MHC class II α chain sequence set forth in SEQ ID NO: 49 can be mutated (e.g., without limitation, to Ala, Trp, Arg, or Gln), or the cysteine ​​in the sequence of the MHC class II α chain of interest, or a portion, fragment, or variant thereof, corresponding to position 70 of the MHC class II α chain sequence set forth in SEQ ID NO: 49 when the sequence of the MHC class II α chain of interest, or a portion, fragment, or variant thereof, is optimally aligned with SEQ ID NO: 49 can be mutated (e.g., without limitation, to alanine or glutamine). Optionally, in some embodiments, the linker includes cysteines at the first three or four residues (and, optionally, in some embodiments, does not include additional cysteines), such as a cysteine ​​at the first, second, third, or fourth residue. In an exemplary embodiment, the linker can include a cysteine ​​at position 3. In another exemplary embodiment, the linker can include a cysteine ​​at position 2.Optionally, in some embodiments, the linker comprises 15 amino acids (such as GCGGSGGGSGGGGS (SEQ ID NO:21)), including a Cys for a disulfide bond to position 101 of the MHC class II α chain (or the position in the sequence of the MHC class II α chain, or portion or fragment or variant thereof, of interest, that corresponds to position 101 of the MHC class II α chain sequence set forth in SEQ ID NO:49, when the MHC class II α chain, or portion or fragment or variant thereof, of interest, is optimally aligned with SEQ ID NO:49). Optionally, in some embodiments, the linker consists essentially of 15 amino acids (such as GCGGSGGGSGGGGS (SEQ ID NO:21)), including a Cys for a disulfide bond to position 101 of the MHC class II α chain (or the position in the sequence of the MHC class II α chain, or portion or fragment or variant thereof, of interest, that corresponds to position 101 of the MHC class II α chain sequence set forth in SEQ ID NO:49, when the MHC class II α chain, or portion or fragment or variant thereof, of interest, is optimally 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 for a disulfide bond to position 101 of the MHC class II α chain (or the position in the sequence of the MHC class II α chain, or portion, fragment, or variant thereof, of interest, that corresponds to position 101 of the MHC class II α chain sequence set forth in SEQ ID NO: 49, when the MHC class II α chain, or portion, fragment, or variant thereof, of interest, is optimally aligned with SEQ ID NO: 49). Optionally, in some embodiments, the MHC class II molecule is an HLA-DQ MHC class II molecule (such as, but not limited to, HLA-DQ2), an HLA-DR MHC class II molecule (such as, but not limited to, HLA-DR2), or an HLA-DP MHC class II molecule.

[0120] In another embodiment, the MHC ligand peptide can include a first cysteine ​​and the MHC class II molecule can include a second cysteine ​​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 anchor position in the MHC ligand peptide can be a cysteine. In another embodiment, the P4 anchor position in the MHC ligand peptide can be a cysteine. In another embodiment, the P6 anchor position in the MHC ligand peptide can be a cysteine. In another embodiment, the P9 anchor position in the MHC ligand peptide can be a cysteine. Optionally, in some embodiments, the cysteine ​​at position 70 of the MHC class II α chain sequence set forth in SEQ ID NO: 49 can be mutated (for example, but not limited to, to Ala, Trp, Arg, or Gln), or the cysteine ​​in the sequence of the MHC class II α chain of interest, or portion or fragment or variant thereof, corresponding to position 70 of the MHC class II α chain sequence set forth in SEQ ID NO: 49 when the sequence of the MHC class II α chain of interest, or portion or fragment or variant thereof, is optimally aligned with SEQ ID NO: 49 can be mutated (for example, but not limited to, to alanine or glutamine). Optionally, in some embodiments, the MHC ligand peptide comprises cysteines at the first three or four residues (and, optionally, in some embodiments, no additional cysteines), such as a cysteine ​​at the first, second, third, or fourth residue. Optionally, in some embodiments, the linker comprises 15 amino acids (such as GCGGGSGGGGSGGGGS (SEQ ID NO: 21)), including a Cys for a disulfide bond to position 101 of the MHC class II α chain (or the position in the sequence of the MHC class II α chain, or portion, fragment, or variant thereof, of interest that corresponds to position 101 of the MHC class II α chain sequence set forth in SEQ ID NO: 49, when the MHC class II α chain, or portion, fragment, or variant thereof, of interest is optimally aligned with SEQ ID NO: 49).Optionally, in some embodiments, the linker consists essentially of 15 amino acids (such as GCGGSGGGSGGGGS (SEQ ID NO:21)), including a Cys for a disulfide bond to position 101 of the MHC class II α chain (or the position in the sequence of the MHC class II α chain, or portion or fragment or variant thereof, of interest, that corresponds to position 101 of the MHC class II α chain sequence set forth in SEQ ID NO:49, when the MHC class II α chain, or portion or fragment or variant thereof, of interest, is optimally aligned with SEQ ID NO:49). Optionally, in some embodiments, the linker consists of 15 amino acids (such as GCGGSGGGSGGGGS (SEQ ID NO:21)), including a Cys for a disulfide bond to position 101 of the MHC class II α chain (or the position in the sequence of the MHC class II α chain, or portion or fragment or variant thereof, of interest, that corresponds to position 101 of the MHC class II α chain sequence set forth in SEQ ID NO:49, when the MHC class II α chain, or portion or fragment or variant thereof, of interest, is optimally aligned with SEQ ID NO:49). Optionally, in some embodiments, the MHC class II molecule is an HLA-DQ MHC class II molecule (for example, but not limited to, HLA-DQ2), an HLA-DR MHC class II molecule (for example, but not limited to, HLA-DR2), or an HLA-DP MHC class II molecule.

[0121] C. Other ingredients In some embodiments of the present invention, a composition comprising a peptide-MHC class II complex may also contain other components. As a non-limiting example, the composition may also contain one or more peptides or one or more other molecules capable of stimulating T helper cells, or one or more immunostimulatory molecules capable of enhancing an immune response. Such T helper cell epitopes or immunostimulatory molecules may be linked (e.g., without limitation, covalently bound) to the peptide-MHC class II complex, or they may not be physically linked to the peptide-MHC class II complex but may be mixed into the composition. In an exemplary embodiment, such T helper cell epitopes or immunostimulatory molecules may be linked (e.g., without limitation, covalently bound) to the peptide-MHC class II complex (e.g., at the C-terminus of the peptide-MHC class II complex). As a non-limiting example, a T helper cell epitope or immunostimulatory molecule can be indirectly or directly linked to the C-terminus of an MHC class II molecule (for example, but not limited to, an MHC class II α chain or a portion, fragment, or variant thereof, and / or an MHC class II β chain or a portion, fragment, or variant thereof). Covalent attachment can be direct or via a linker, such as a peptide linker. In some embodiments, non-limiting examples of linkers suitable for use in the disclosed peptide-MHC class II complexes are described elsewhere herein.

[0122] As a non-limiting example of some embodiments, T helper cell epitopes suitable for use in the disclosed peptide-MHC class II complexes are pan-DR (human MHC class II) binding epitopes (PADRE) peptides or molecules designed based on their binding activity to most HLA-DR (human MHC class II) molecules. PADREs are "pan-DR binding epitopes" and bind to the murine MHC IA, as described in Alexander et al. (2000) J. Immunol. 164(3):1625-1633, which is incorporated herein by reference in its entirety for all purposes. bA mouse MHC-II binding sequence used to enhance immune responses based on the provision of a "universal" MHC-II epitope presented by a haplotype. It can be fused to the terminus of an antigen used for immunization, and its uptake by antigen-presenting cells and presentation by MHC-II improves the overall immune response. See, e.g., 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 be useful in generating a variety of immune responses to antigens. In some embodiments, the PADRE peptide can comprise AKFVAAWTLKAAA (SEQ ID NO: 25). In some embodiments, the PADRE peptide can consist essentially of AKFVAAWTLKAAA (SEQ ID NO: 25). In some embodiments, the PADRE peptide can consist of AKFVAAWTLKAAA (SEQ ID NO: 25).

[0123] Similar to PADRE, peptides derived from lymphocytic choriomeningitis virus (LCMV) (e.g., LCMV glycoprotein (GP), nucleoprotein (NP), or zinc-binding protein (Z)) are alternative MHC-II binding small polypeptides that can be used to enhance immune responses. In some embodiments, such peptides can be used in addition to or as an alternative to PADRE. In some embodiments, the LCMV peptide used can be an LCMV-specific MHC class II restricted CD4+ T cell epitope. In some embodiments, the LCMV peptide used can comprise one or more of the following sequences: TMFEALPHIIDEVIN (epitope GP) 6-20 , SEQ ID NO: 26), GIKAVYNFATCGIFA (epitope GP31-45, SEQ ID NO: 27), DIYKGVYQFKSVEFD (epitope GP 66-80 , SEQ ID NO: 28), TSAFNKKTFDHTLMS (epitope GP 126-140, SEQ ID NO: 29), DAQSAQSQCRTFRGR (epitope 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 (epitope GP 409-423 , SEQ ID NO: 33), EVKSFQWTQALRREL (epitope NP 6-20 , SEQ ID NO: 34), KNVLKVGRLSAEELM (epitope NP 86-100 , SEQ ID NO: 35), SERPQASGVYMGNLT (epitope NP 116-130 , SEQ ID NO: 36), PSLTMACMAKQSQTP (epitope NP 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 (epitope Z 31-45 , SEQ ID NO: 41). In some embodiments, the LCMV peptide used can consist essentially of one or more of the following sequences: TMFEALPHIIDEVIN (epitope GP 6-20 , SEQ ID NO: 26), GIKAVYNFATCGIFA (epitope GP31-45, SEQ ID NO: 27), DIYKGVYQFKSVEFD (epitope GP 66-80 , SEQ ID NO: 28), TSAFNKKTFDHTLMS (epitope GP 126-140 , SEQ ID NO: 29), DAQSAQSQCRTFRGR (epitope 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 (epitope GP 409-423 , SEQ ID NO: 33), EVKSFQWTQALRREL (epitope NP 6-20, SEQ ID NO: 34), KNVLKVGRLSAEELM (epitope NP 86-100 , SEQ ID NO: 35), SERPQASGVYMGNLT (epitope NP 116-130 , SEQ ID NO: 36), PSLTMACMAKQSQTP (epitope NP 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 (epitope Z 31-45 , SEQ ID NO: 41). In some embodiments, the LCMV peptide used can consist of one or more of the following sequences: TMFEALPHIIDEVIN (epitope GP 6-20 , SEQ ID NO: 26), GIKAVYNFATCGIFA (epitope GP31-45, SEQ ID NO: 27), DIYKGVYQFKSVEFD (epitope GP 66-80 , SEQ ID NO: 28), TSAFNKKTFDHTLMS (epitope GP 126-140 , SEQ ID NO: 29), DAQSAQSQCRTFRGR (epitope 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 (epitope GP 409-423 , SEQ ID NO: 33), EVKSFQWTQALRREL (epitope NP 6-20 , SEQ ID NO: 34), KNVLKVGRLSAEELM (epitope NP 86-100 , SEQ ID NO: 35), SERPQASGVYMGNLT (epitope NP 116-130 , SEQ ID NO: 36), PSLTMACMAKQSQTP (epitope NP 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 (epitope Z 31-45 , SEQ ID NO: 41). See, e.g., Botten et al. (2010) Microbiol. Mol. Biol. Rev. 74(2):157-170 and Mothe et al. (2007) J. Immunol. 179(2):1058-1067, each of which is incorporated by reference in its entirety for all purposes. In some embodiments, the peptide can comprise SERPQASGVYMGNLT (SEQ ID NO: 36). In some embodiments, the peptide can consist essentially of SERPQASGVYMGNLT (SEQ ID NO: 36). In some embodiments, the peptide can consist of SERPQASGVYMGNLT (SEQ ID NO: 36).

[0124] In some embodiments, one T cell epitope (e.g., an LCMV peptide) is added to a composition comprising a peptide-MHC class II complex. In some embodiments, multiple T cell epitopes (e.g., without limitation, two T cell epitopes, three T cell epitopes, four T cell epitopes, five T cell epitopes, six T cell epitopes, seven T cell epitopes, eight T cell epitopes, nine T cell epitopes, or ten T cell epitopes) are added to a composition comprising a peptide-MHC class II complex. In some embodiments, about one to about ten T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about two to about ten T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about three to about ten T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about 4 to about 10 T cell epitopes can be added to a composition comprising peptide-MHC class II complexes. In some embodiments, about 5 to about 10 T cell epitopes can be added to a composition comprising peptide-MHC class II complexes. In some embodiments, about 6 to about 10 T cell epitopes can be added to a composition comprising peptide-MHC class II complexes. In some embodiments, about 7 to about 10 T cell epitopes can be added to a composition comprising peptide-MHC class II complexes. In some embodiments, about 8 to about 10 T cell epitopes can be added to a composition comprising peptide-MHC class II complexes. In some embodiments, about 9 to about 10 T cell epitopes can be added to a composition comprising peptide-MHC class II complexes. In some embodiments, about 1 to about 9 T cell epitopes can be added to a composition comprising peptide-MHC class II complexes. In some embodiments, about 1 to about 8 T cell epitopes can be added to a composition comprising a peptide-MHC class II complex.In some embodiments, about one to about seven T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about one to about six T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about one to about five T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about one to about four T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about one to about three T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about one to about two T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about two to about nine T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about three to about eight T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about four to about seven T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about four to about six T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, about two to about four T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, each of the multiple T cell epitopes can be, but is not limited to, an LCMV peptide, and each LCMV peptide can comprise any of the sequences described above. In some embodiments, each of the multiple T cell epitopes can be, but is not limited to, an LCMV peptide, and each LCMV peptide can consist essentially of any of the sequences described above. In some embodiments, each of the multiple T cell epitopes can be, but is not limited to, an LCMV peptide, and each LCMV peptide can consist of any of the sequences described above.

[0125] In some embodiments, one T cell epitope (e.g., an LCMV peptide) is added to a composition comprising a peptide-MHC class II complex. In some embodiments, multiple T cell epitopes (e.g., without limitation, two T cell epitopes, three T cell epitopes, four T cell epitopes, five T cell epitopes, six T cell epitopes, seven T cell epitopes, eight T cell epitopes, nine T cell epitopes, or ten T cell epitopes) are added to a composition comprising a peptide-MHC class II complex. In some embodiments, between one and ten T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, between two and ten T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, between three and ten T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, between 4 and 10 T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, between 5 and 10 T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, between 6 and 10 T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, between 7 and 10 T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, between 8 and 10 T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, between 9 and 10 T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, between 1 and 9 T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, between 1 and 8 T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, between one and seven T cell epitopes can be added to a composition comprising a peptide-MHC class II complex.In some embodiments, one to six T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, one to five T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, one to four T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, one to three T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, one to two T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, two to nine T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, three to eight T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, four to seven T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, four to six T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, two to four T cell epitopes can be added to a composition comprising a peptide-MHC class II complex. In some embodiments, each of the multiple T cell epitopes can be, but is not limited to, an LCMV peptide, and each LCMV peptide can comprise any of the sequences described above. In some embodiments, each of the multiple T cell epitopes can be, but is not limited to, an LCMV peptide, and each LCMV peptide can consist essentially of any of the sequences described above. In some embodiments, each of the multiple T cell epitopes can be, but is not limited to, an LCMV peptide, and each LCMV peptide can consist of any of the sequences described above.

[0126] In some embodiments, other peptides or molecules can also be used in the compositions provided herein to improve immune responses. Non-limiting examples include immunostimulants such as keyhole limpet hemocyanin (KLH) or polypeptides that are cross-presented by multiple haplotypes of the immunized host (e.g., but not limited to, mouse or rat). Such peptides can be, for example, fused to peptide-MHC class II complexes or can be molecules provided separately (e.g., but not limited to, mixed in the same composition).

[0127] In some embodiments, peptide or other tags can also be used in the compositions, for example, to facilitate purification. In some embodiments, non-limiting examples of tags suitable for use in the disclosed peptide-MHC class II complexes 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 9-amino acid 1D4 epitope derived from the C-terminus of bovine rhodopsin). In some embodiments, the sequence of the BirA tag can comprise GLNDIFEAQKIEWHE (SEQ ID NO: 42). In some embodiments, the sequence of the BirA tag can consist essentially of GLNDIFEAQKIEWHE (SEQ ID NO: 42). In some embodiments, the sequence of the BirA tag can consist of GLNDIFEAQKIEWHE (SEQ ID NO: 42). In some embodiments, the sequence of the mmH tag can include EQKLISEEDLEQKLISEEDLHHHHHH (SEQ ID NO: 43) or EQKLISEEDLGGEQKLISEEDLHHHHHH (SEQ ID NO: 48). In some embodiments, the sequence of the mmH tag can consist essentially of EQKLISEEDLEQKLISEEDLHHHHHH (SEQ ID NO: 43) or EQKLISEEDLGGEQKLISEEDLHHHHHH (SEQ ID NO: 48). In some embodiments, the sequence of the mmH tag can consist of EQKLISEEDLEQKLISEEDLHHHHHH (SEQ ID NO: 43) or EQKLISEEDLGGEQKLISEEDLHHHHHH (SEQ ID NO: 48).

[0128] III. Nucleic Acids Encoding Peptide-MHC Class II Complexes Nucleic acids encoding the peptide-MHC class II complexes disclosed in some embodiments of the present invention are also provided. Such nucleic acids can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or hybrids or derivatives of either DNA or RNA. Optionally, in some embodiments, the nucleic acid encoding the peptide-MHC class II can be codon-optimized for efficient translation into protein in a particular cell or organism. As a non-limiting example, the nucleic acid encoding the peptide-MHC class II can be modified to use alternative codons more frequently used in mammalian cells, rodent cells, mouse cells, rat cells, or any other host cell of interest, compared to the naturally occurring polynucleotide sequence. Any portion or fragment of the nucleic acid molecule can be produced by: (1) isolating the molecule from its natural environment; (2) using recombinant DNA technology (e.g., PCR amplification or cloning); or (3) using chemical synthesis. The nucleic acid encoding the peptide-MHC class II complex can include modifications for improved stability or reduced immunogenicity. Non-limiting examples of modifications include: (1) alteration or substitution of one or both of the non-linked phosphate oxygens and / or one or more of the linking phosphate oxygens in the phosphodiester backbone linkages; (2) alteration or substitution of a component of the ribose sugar, such as alteration or substitution of the 2' hydroxyl of the ribose sugar; (3) replacement of a phosphate moiety with a dephosphorylatable linker; (4) alteration or substitution of a naturally occurring nucleobase; (5) substitution or modification of the ribose-phosphate backbone; (6) modification of the 3' or 5' end of the oligonucleotide (e.g., but not limited to, removal, modification, or substitution of a terminal phosphate group or attachment of a moiety); and (7) sugar modification.

[0129] In some embodiments, the nucleic acid can be in the form of an expression construct, as defined elsewhere herein. As a non-limiting example, the nucleic acid can include regulatory regions (e.g., transcriptional or translational control regions) that control expression of the nucleic acid molecule, full-length or partial coding regions, and combinations thereof. As a non-limiting example, the nucleic acid can be operably linked to a promoter active in a cell or organism of interest. Promoters that can be used in such expression constructs include promoters active in one or more eukaryotic cells, such as mammalian cells (e.g., non-human mammalian cells or human cells), including, for example, rodent cells (e.g., but not limited to, mouse cells, or rat cells). Such promoters can be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters.

[0130] In some embodiments, nucleic acids can include functional equivalents of naturally occurring nucleic acid molecules, such as, but not limited to, naturally occurring allelic variants and modified nucleic acid molecules that encode MHC molecules or peptides, in which nucleotides have been inserted, deleted, substituted, and / or inverted in such a way that such modifications do not substantially interfere with the ability of the nucleic acid molecule to encode a protein capable of forming a composition comprising a peptide-MHC class II complex (e.g., capable of being recognized by a T cell receptor) as described elsewhere herein.

[0131] IV. METHODS OF USE OF PEPTIDE-MHC CLASS II COMPLEXES Also provided are methods of eliciting an immune response in a subject, comprising administering to the subject an effective amount of a composition comprising a peptide-MHC class II complex described elsewhere herein.

[0132] In some embodiments of the present 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, oxen, deer, bison, sheep, rabbits, rodents (for example, but not limited to, mice, rats, hamsters, and guinea pigs), and livestock (for example, but not limited to, bovine species such as cows and steers, ovine species such as sheep and goats, and porcine species such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domestic and agricultural animals are also included. The term "non-human animal" excludes humans. Specific, non-limiting examples of non-human animals include rodents such as mice and rats.

[0133] The term administration refers to the administration of a composition to a subject or system (e.g., without limitation, a cell, organ, tissue, organism, or associated component or set of components thereof). The route of administration can vary depending, for example, on the subject or system to which the composition is administered, the nature of the composition, the purpose of the administration, etc. The term "administration" or "administering" is intended to include routes by which peptide-MHC class II complexes are introduced into a subject to perform their intended function (e.g., inducing or modulating an immune response). In some embodiments, non-limiting examples of routes of administration that can be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal), oral, inhalation, rectal, and transdermal. By way of non-limiting example, administration to a subject (e.g., including but not limited to, administration to a human or rodent) can be bronchial (including via bronchial instillation), buccal, enteral, intercutaneous, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intracerebroventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including intratracheal instillation), transdermal, intravaginal, and / or intravitreal. The peptide-MHC class II complexes can be administered in tablet or capsule form (e.g., but not limited to, via injection, inhalation, eye lotion, ointment, suppository, etc.), topically via lotion or ointment, or rectally via suppository. Administration can be via bolus or continuous infusion. Administration can include intermittent or continuous dosing (e.g., but not limited to, via perfusion) for at least a selected period of time. Depending on the route of administration, the peptide-MHC class II complexes can be coated or otherwise disposed with a selected material to protect the peptides from natural conditions that may adversely affect their ability to perform their intended function. The peptide-MHC class II complexes can be administered alone or in combination with another agent (e.g., an immunostimulant), a pharmaceutically acceptable carrier, or both. The peptide-MHC class II complexes can be administered before, simultaneously with, or after the administration of other agents. Furthermore, the peptide-MHC class II complexes can be administered in a proform that is converted in vivo to its active or more active metabolite.

[0134] Also provided are methods of making antigen-binding proteins, comprising immunizing a non-human animal with a peptide-MHC class II complex described elsewhere herein, allowing the non-human animal to mount 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 comprise 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 can specifically bind to an epitope on the peptide-MHC class II complex (e.g., with an equilibrium dissociation constant (KD) in the micromolar, nanomolar, or picomolar range). In some embodiments, the antigen-binding protein can be a therapeutic antigen-binding protein or antibody (e.g., for use in a patient).

[0135] In one exemplary embodiment, the cell is a B cell isolated from a non-human animal, and the method further comprises identifying nucleic acid sequences of immunoglobulin heavy and light chain variable regions that encode immunoglobulin heavy and light chain variable domains that, when paired, specifically bind to a peptide-MHC class II complex. Such a method can further comprise expressing the nucleic acid sequences in an expression system suitable for expressing the antigen binding protein to form an antigen binding protein comprising a dimer of the heavy and light chain variable domains that binds to the peptide-MHC class II complex.

[0136] In another embodiment, the method includes isolating nucleic acid from the non-human animal and obtaining an immunoglobulin heavy chain variable region sequence and / or an immunoglobulin light chain variable region sequence encoding an immunoglobulin heavy chain variable domain and / or an immunoglobulin light chain variable domain, respectively, of an antibody that specifically binds to a peptide-MHC class II complex. Such a method can further include using the immunoglobulin heavy chain variable region sequence and / or the immunoglobulin light chain variable region sequence to produce an antibody that binds to a peptide-MHC class II complex.

[0137] In some embodiments of the method, cells (such as B cells) are collected from a non-human animal (for example, but not limited to, from the spleen or lymph nodes). The cells may be fused with a myeloma cell line to prepare immortalized hybridoma cell lines, which are screened and selected to identify hybridoma cell lines that produce antibodies containing a hybrid heavy chain specific for the antigen used for immunization.

[0138] In some embodiments of the method, immunization involves priming (e.g., without limitation, administering) the non-human animal with a peptide-MHC class II complex, allowing the non-human animal to rest for a period of time, and re-immunizing the non-human animal with the peptide-MHC class II complex (e.g., without limitation, to boost the immune response). In some embodiments of the method, the method involves immunizing and / or boosting the non-human animal simultaneously with a helper T cell epitope, such as, without limitation, a pan-DR T helper epitope (PADRE). See, e.g., U.S. Patent No. 6,413,935 and Alexander et al. (1994) Immunity 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 comprises priming the non-human animal with a peptide-MHC class II complex and boosting 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 comprises both priming and boosting the non-human animal with a peptide-MHC class II complex linked to a helper T cell epitope. In methods involving priming and / or boosting with PADRE, the non-human animal can be a mouse comprising a C57 / BL6 genetic background. In some embodiments, the C57BL strain of mice can be C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KalLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10C, ​​and C57BL / Ola, or a hybrid of the aforementioned C57BL / 6 strain with another strain (e.g., 129, BALB, etc.). In some embodiments of the method, the period between priming the non-human animal and boosting 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.

[0139] In some embodiments of the method, the non-human animal can comprise a human or humanized immunoglobulin heavy and / or light chain locus, thereby providing the non-human animal with a human or humanized antigen-binding protein-binding domain (e.g., a human or humanized variable domain) comprising a human or humanized antigen. Immunoglobulin loci comprising human variable region gene segments are known in the art, and include, by way of non-limiting example, 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, 7,910,798, and 8,233. Nos. 2,449, 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, 9,580,491 and 9,47 No. 5,559, as well as U.S. Patent Application Publication Nos. 20100146647, 20110195454, 20130167256, 20130219535, 20130326647, 20130096287, and 20150113668, which are incorporated herein by reference in their entireties for all purposes. These and other related art techniques may be outlined in PCT Publication Nos. 2007 / 117410, 2008 / 151081, 2009 / 157771, 2010 / 039900, 2011 / 004192, 2011 / 123708, and 2014 / 093908, each of which is incorporated herein by reference in its entirety for all purposes.As a non-limiting example, a non-human animal can comprise unrearranged or rearranged human or humanized immunoglobulin heavy chain and / or unrearranged or rearranged human or humanized immunoglobulin light chain loci in its genome, thereby enabling the non-human animal to provide human or humanized antigen binding proteins that comprise human or humanized antigen binding domains (e.g., human or humanized immunoglobulin variable domains), optionally, in some embodiments, at least one of the human or humanized immunoglobulin heavy chain loci and / or human or humanized immunoglobulin light chain loci is unrearranged.

[0140] In some embodiments, such methods can further comprise cloning a nucleotide sequence encoding a human heavy 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, or independently, be a universal light chain variable domain) in frame with a gene encoding a human heavy chain constant region (CH) or light chain constant region (CL) to form a human binding protein sequence, and expressing the human binding protein sequence in a suitable cell.

[0141] Also provided is a method for identifying T cells having specificity for an antigenic peptide or peptide-MHC class II complex, the method comprising immunizing a non-human animal with a peptide-MHC class II complex as described elsewhere herein, mounting 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.

[0142] Also provided are methods for generating nucleic acid sequences encoding TCR variable domains (e.g., TCR α and / or β variable domains). In some embodiments, such methods can include immunizing a non-human animal with a peptide-MHC class II complex, as described elsewhere herein, allowing the non-human animal to mount an immune response to the peptide-MHC class II complex, and obtaining therefrom 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 can further include generating a nucleic acid sequence encoding a TCR variable domain operably linked to a TCR constant region, isolating a T cell from the non-human animal herein, and obtaining therefrom a nucleic acid sequence encoding a TCR variable domain linked to a TCR constant region. In some embodiments, the non-human animal can comprise a humanized T cell receptor variable gene locus, and the method can include determining the nucleic acid sequence of a human TCR variable region expressed by the T cell, and cloning the human TCR variable region into a nucleotide construct comprising the nucleic acid sequence of the human TCR constant region, such that the human TCR variable region is operably linked to the human TCR constant region. Optionally, in some embodiments, the method can further include expressing from the construct (e.g., in the cell) a human TCR specific for the peptide or peptide-MHC class II complex.

[0143] Also provided are methods for generating a T cell receptor (TCR) specific for an antigenic peptide or peptide-MHC class II complex, the methods comprising immunizing a non-human animal with a peptide-MHC class II complex as described elsewhere herein, mounting 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 can further comprise determining the nucleic acid sequence of a TCR variable region expressed by the T cell, cloning the TCR variable region into a nucleotide construct comprising the nucleic acid sequence of the TCR constant region such that the TCR variable region is operably linked to the TCR constant region, and optionally expressing (e.g., expressing intracellularly) the TCR specific for the peptide or peptide-MHC class II complex from the construct. In some embodiments, the non-human animal can comprise a humanized T cell receptor variable gene locus, and the method can include determining the nucleic acid sequence of a human TCR variable region expressed by the T cell, and cloning the human TCR variable region into a nucleotide construct comprising the nucleic acid sequence of a human TCR constant region, such that the human TCR variable region is operably linked to the human TCR constant region. Optionally, in some embodiments, the method can further include expressing (e.g., in a cell) a human TCR specific for the peptide or peptide-MHC class II complex from the construct.

[0144] In some embodiments, the identified T cells or TCRs specific for the antigenic peptide or peptide-MHC class II complex can be used for treatment (e.g., adoptive T cell therapy) in a subject. In some embodiments, for example, such methods can include immunizing a non-human animal with a peptide-MHC class II complex as described elsewhere herein, mounting 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 cell, cloning the nucleic acid sequence of the TCR into an expression vector (e.g., a retroviral vector), introducing the vector into T cells from the subject such that the T cell expresses an antigen-specific T cell receptor, and infusing the T cell into the subject. In some embodiments, the antigen-specific T cell population is expanded before infusion into the subject. In some embodiments, the subject's immune cell population is immunodepleted before infusion of the antigen-specific T cells.

[0145] In some embodiments of the method, the non-human animal can express a humanized T cell receptor. See, e.g., U.S. Patent No. 9,113,616, 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 can comprise a humanized T cell receptor variable locus. As a non-limiting example, the non-human animal can express humanized TCR alpha and beta polypeptides (and / or humanized TCR delta and TCR gamma polypeptides). In one embodiment, the non-human animal comprises an unrearranged human TCR variable locus in its genome.

[0146] In some embodiments of the method, the non-human animal is tolerized to at least one empty human or humanized MHC class II molecule, or at least the empty human peptide-binding groove thereof, when it forms a complex with an antigenic (e.g., without limitation, a heterologous) peptide, but is capable of producing an antigen-binding protein to the human(ized) MHC molecule (e.g., without limitation, an antigen-binding protein comprising a human or humanized variable domain). In some embodiments of the method, tolerization of the non-human animal to the empty human(ized) MHC class II molecule is achieved by genetically modifying the non-human animal to include in its genome a nucleotide sequence encoding the human(ized) MHC molecule, or at least the human peptide-binding groove thereof, such that the non-human animal expresses the human(ized) MHC molecule, or at least the human peptide-binding groove thereof, as an empty human(ized) MHC molecule, or the empty human peptide-binding groove thereof. The same animal genetically modified to contain nucleotides encoding human(ized) MHC molecules can be further modified to contain humanized immunoglobulin heavy and / or light chain loci expressing human or humanized antigen-binding proteins (e.g., without limitation, antigen-binding proteins having human or humanized variable domains) and / or humanized T cell receptor variable gene 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, e.g., US 2019 / 0292263, incorporated herein by reference in its entirety for all purposes. The MHC II nucleotide sequence can encode an MHC II protein that is fully human (e.g., without limitation, a human HLA class II molecule) or a humanized MHC class II protein that is partially human and partially non-human (e.g., including without limitation, a chimeric human / non-human MHC II protein, e.g., a chimeric human / non-human MHC IIα and β polypeptide).Genetically modified non-human animals comprising in their genome (e.g., not limited to the endogenous locus) a nucleotide sequence encoding a humanized (e.g., chimeric human / non-human) MHC II polypeptide are disclosed in U.S. Pat. Nos. 8,847,005 and 9,043,996, each of which is incorporated herein by reference in its entirety for all purposes.

[0147] While tolerization of chimeric human / non-human MHC molecules to human peptide-binding domains can be achieved by expression from endogenous MHC loci, in some embodiments, such tolerization also occurs in non-human animals that express human MHC class II molecules (or functional peptide-binding domains thereof) from ectopic loci. Furthermore, in some embodiments, non-human animals that express and are tolerized to empty human MHC class II molecules (or empty peptide-binding domains thereof) from ectopic loci can generate a specific immune response against the human HLA molecule (or peptide-binding domains thereof or derivatives thereof) from which the expressed human MHC class II molecule is derived when the non-human animal is immunized with a human HLA molecule (or peptide-binding domains thereof and / or derivatives thereof) complexed with an antigenic peptide (e.g., but not limited to, a peptide xenogeneic to the non-human animal). Without wishing to be bound by theory, it is believed that tolerization of non-human animals occurs upon expression of human or humanized MHC class II molecules. Thus, it is not necessary for the human or humanized MHC class II molecule to be expressed from an endogenous locus.

[0148] In some embodiments, such methods can further include breaking tolerance to endogenous peptides. Immunization of non-human animals (e.g., 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 relies on sequence differences between the non-human animal's endogenous proteins and the presented foreign proteins, allowing the non-human animal's immune system to recognize the peptide-MHC class II complex as non-self (i.e., foreign). The generation of antibodies and T cells / TCRs against peptide-MHC class II complexes that are highly homologous to self-peptide-MHC class II complexes can be difficult due to immune tolerance to self-peptide-MHC class II complexes. Methods for breaking tolerance to self-peptides that are homologous to a peptide of interest are well known. See, for example, U.S. Patent Application Publication No. 2017 / 0332610, which is incorporated herein by reference in its entirety for all purposes. In some embodiments of such methods, the method of breaking tolerance to an endogenous peptide comprises modifying a non-human animal herein to contain a deletion (e.g., a knockout mutation) of a self-peptide that has high homology to the peptide of interest.

[0149] The non-human animals in the methods disclosed in some embodiments herein can include any type of non-human animal, such as a mammal. Mammals include, for example, humans, non-human mammals, non-human primates, monkeys, apes, cats, dogs, horses, oxen, 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, bovine species such as cows and steers, ovine species such as sheep and goats, and porcine species such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domestic and agricultural animals are also included. The term "non-human animal" excludes humans. Specific, non-limiting examples of non-human animals include rodents such as mice and rats.

[0150] All patent applications, websites, other publications, accession numbers, etc., cited above or below are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other. Although the invention has been described in some detail through diagrams and examples for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0151] A brief description of arrays The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5'-end of the sequence and proceeding forward (i.e., left to right on each line) to the 3'-end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the shown strand. Where a nucleotide sequence encoding an amino acid sequence is provided, it is understood that codon-degenerate variants thereof that encode the same amino acid sequence are also provided. Where a DNA sequence encoding an amino acid sequence is provided, it is understood that an RNA sequence that encodes the same amino acid sequence is also provided (by substituting thymine with uracil). The amino acid sequences follow the standard convention of beginning at the amino-terminus of the sequence and proceeding forward (i.e., left to right on each line) to the carboxy-terminus. [Table 1-1] [Table 1-2] [Example]

[0152] Example 1. Design of peptide-MHC II protein constructs Examples of soluble peptide-MHC I protein constructs have been previously described. Such constructs can be used for a variety of applications, such as immunizing rodents with VELOCIMMUNE® to generate anti-peptide in-growth antibodies. This example describes the design of peptide-MHC II protein constructs in which the α and β chains of the MHC II molecule are anchored together. These can be used for a variety of applications, such as generating soluble MHC II constructs to function as immunogens or membrane-anchored MHC II proteins for other applications, including recruiting T cells expressing MHC class II peptide-specific T cell receptors (TCRs). Soluble or membrane-anchored MHC II proteins can also be used for specific targeting of T cells expressing MHC class II peptide-specific T cell receptors (TCRs) to modulate T cell activity or viability in various disease conditions.

[0153] Various soluble peptide-MHC II constructs were designed, as shown in Figure 1. A description of the soluble peptide-MHC II constructs is provided in Table 2. Some constructs include E. coli biotin ligase (BirA) and myc-myc-histidine (mmH) tags, although 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. An alignment of the full-length DQ2 α-chain segment used in the constructs (including the C70Q mutation or both the R101C and C70A mutations) is shown in Figure 2. For the C70 mutation, numbering may vary based on the reference sequence or the signal sequence selected for a given construct. C70 is the position within the full-length HLA-II DQα1 chain sequence designated as UniProt accession number P01909-1 (SEQ ID NO: 49). A version of the full-length HLA-II DQα1 chain sequence with the C70Q mutation is set forth in SEQ ID NO:55, and a version of the full-length HLA-II DQα1 chain sequence with both the R101C and C70A mutations is set forth in SEQ ID NO:54. The portions of the full-length HLA-II DQα1 chain included in the soluble HLA-DQ2 constructs tested below included residues 24-216 of SEQ ID NO:49 (no R101 or C70 mutations), SEQ ID NO:55 (C70Q mutation), or SEQ ID NO:54 (R101C and C70A mutations). A version of the full-length HLA-II DQβ1 chain sequence is designated NCBI accession number NP_001230891.1 (SEQ ID NO:50). The portions of the full-length HLA-II DQβ1 chain included in the soluble HLA-DQ2 constructs tested below included residues 33-230 of SEQ ID NO:50. An alignment of full-length α chain segments from different HLA class II alleles is shown in FIG. [Table 2]

[0154] Good yields were observed for constructs A through C. Proteins were purified using standard procedures, including affinity and size-exclusion chromatography. The final protein mass obtained after purification was determined by UV absorbance and the calculated extinction coefficient based on the amino acid composition of the protein. Production yields were calculated by dividing the mass of purified protein by the volume of medium. Construct A, when covalently coupled to the QLQPFPQPELPY (SEQ ID NO: 44, "QLQ" peptide) peptide, gave a purified yield of 14 mg / L. Construct C, when covalently coupled to the QLQ peptide (SEQ ID NO: 44), gave a purified yield of 2.4 mg / L. Construct B, when separately covalently coupled to the QLQ peptide (SEQ ID NO: 44), FPQPEQPFPWQP (SEQ ID NO: 45; "FPQ" peptide), and PQPELPYPQPQL (SEQ ID NO: 46; "PQP" peptide), gave purified yields of 204 mg / L, 36 mg / L, and 0.9 mg / L, respectively. A summary of the results is shown in Table 3.

[0155] Measurable yields of soluble protein were consistently obtained with peptide-MHC II constructs containing: (1) a C-terminal jun / fos zipper connected to the α and β chains of MHC II by a SGGGGG (SEQ ID NO: 1) linker; (2) the R101C mutation introduced into the α chain of MHC II; (3) a linker at the N-terminus of the β-chain connected to the peptide (the linker contains an additional Cys mutation, allowing disulfide bond formation between the Cys of the linker and the R101C mutation introduced into the α-chain of MHC II), and (4) removal of the unpaired Cys in the α chain (C70A mutation);

[0156] The unpaired Cys in DQA1*0501 is replaced with Trp, Arg, or Gln in the closest MHC sequence from other species based on sequence alignment, so mutations to these residues can be used instead. [Table 3]

[0157] MHC constructs were tested for their ability to bind antibodies against MHC class II proteins via two different Biacore assays. For both assay formats, the instrument used was an Octet HTX, the chip type was an Octet biosensor coated with anti-mouse or anti-human Fc, the assay was run at 25°C, the running buffer was HBS-ET + 1 mg / mL BSA, the capture mixing speed and time was 1000 rpm and 1 minute, and the sample injection mixing speed and time was 1000 rpm and 2 minutes.

[0158] Construct C was analyzed to verify binding to monoclonal antibodies. In the first experiment, approximately 0.8 nm of a pan-class II anti-HLA mAb or anti-DR / DQ mAb was captured by immersing an anti-mFc-coated octet biosensor for 1 minute in a well containing 100 nM of mAb. The mAb-captured sensor was then submerged in a well containing 200 nM of construct C. As shown in Figure 4 and Table 4, soluble construct C bound to both anti-class II monoclonal antibodies captured on the anti-mFc sensor surface but not to the isotype control mAb. In the second experiment, approximately 1 nm of construct C was captured by immersing an anti-hFc-coated octet biosensor for 1 minute in a well containing 200 nM of construct C. The construct C-captured sensor was then submerged in a well containing 100 nM of a pan-class II anti-HLA mAb or anti-DR / DQ mAb. As shown in Figure 5 and Table 5, soluble construct C captured on the anti-hFc sensor surface bound both anti-class II monoclonal antibodies but not the isotype control mAb. The pan-class II anti-HLA antibody bound only to properly folded HLA proteins, verifying the conformational integrity of the produced and purified proteins. [Table 4] [Table 5]

[0159] Construct B is analyzed to verify binding to the monoclonal antibodies. In the first experiment, approximately 0.8 nm of a pan-class II anti-HLA mAb or anti-DR / DQ mAb is captured by immersing an anti-mFc-coated octet biosensor in a well containing 100 nM of mAb for 1 minute. The mAb-captured sensor is then submerged in a well containing 200 nM of construct B. Soluble construct B binds to both anti-class II monoclonal antibodies captured on the anti-mFc sensor surface, but not to the isotype control mAb. In the second experiment, approximately 1 nm of construct B is captured by immersing an anti-hFc-coated octet biosensor in a well containing 200 nM of construct B for 1 minute. The construct B-captured sensor is then submerged in a well containing 100 nM of a pan-class II anti-HLA mAb or anti-DR / DQ mAb. Soluble construct B captured on the anti-hFc sensor surface binds both anti-class II monoclonal antibodies but not the isotype control mAb. The pan-class II anti-HLA antibody binds only to properly folded HLA proteins, verifying the conformational integrity of the produced and purified proteins.

[0160] Example 2. Tolerization of mice Mice tolerized to empty MHC class II molecules are generated or provided, but the MHC class II molecules are not derived from a mouse (e.g., but not limited to, a human). For example, a first mouse expressing an MHC class II molecule derived from a corresponding endogenous locus or a locus other than the corresponding endogenous locus (e.g., but not limited to, the ROSA26 locus) is tolerized to empty MHC class II molecules. These tolerized mice are then injected with an immunogen (e.g., an MHC class II molecule containing an immunogenic peptide in its groove, such as construct A, construct B, or construct C from Example 1). These immunized mice produce specific antibody titers against this particular immunogen compared to mice not tolerized to empty MHC class II molecules. Alternatively, mice not tolerized and immunized with the target MHC class II molecule produce antibodies that not only recognize the immunogenic peptide but also the MHC class II molecule. Thus, tolerized mice immunized with the MHC class II molecules described herein are capable of generating an antigen-specific immune response without generating antibodies directed solely against the MHC class II molecules.

[0161] Example 3. Immunization of tolerized mice with MHC protein constructs The peptides to be tethered to the HLA-DQB chain as DNA and soluble dimeric proteins are selected for immunization and screening. Schematic diagrams of examples of constructs containing peptides tethered to the HLA-DQB chain for immunization and screening are shown in Figures 6A and 6B.

[0162] Mice (e.g., mice containing humanized immunoglobulin heavy and / or light chain variable region loci) are immunized with a peptide-MHC (pMHC) complex of interest comprising a peptide antigenic to the mouse and a human or humanized MHC class II molecule to which the mouse is to be tolerized. The mice are additionally and optionally boosted with a pMHC complex of interest, which booster is optionally linked to a helper T cell epitope. Antibodies (e.g., human or humanized antibodies expressed from the humanized immunoglobulin heavy and / or light chain loci) are isolated from the immunized mice and tested for binding specificity to the pMHC complex.

[0163] Test mice are provided that are tolerized to human MHC II molecules and comprise nucleotide sequences encoding a humanized immunoglobulin heavy chain locus (see, e.g., Macdonald (2014) Proc. Natl. Acad. Sci. USA 111:5147-5152, which is incorporated by reference in its entirety for all purposes), and a humanized common light chain locus (see, e.g., U.S. Pat. Nos. 10,143,186, 10,130,081, and 9,969,814; U.S. Patent Application Publication Nos. 2012 / 0021409, 2012 / 0192300, 2013 / 0045492, 2013 / 0185821, 2013 / 0302836, and 2015 / 0313193, each of which is incorporated by reference in its entirety for all purposes). These test mice, as well as non-tolerized control mice containing a functional (e.g., murine) ADAM6 gene (see, e.g., U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated by reference in its entirety for all purposes) and humanized immunoglobulin heavy and light chain loci, are immunized with pMHC complexes containing heterologous peptide inclusions presented in the context of HLA-DQ molecules, either administered as protein immunogens or DNA encoding the pMHC complexes. Mice are boosted at various time intervals via various routes using pMHC complex immunogens containing standard adjuvants or pMHC complex immunogens linked to a T helper pan-DR epitope (PADRE) peptide. Pre-immune serum is collected from the mice before the start of immunization. Mice are bled periodically, and antiserum titers are analyzed for each antigen.

[0164] Antibody titers in serum against an irrelevant antigen (i.e., an antigen that the mice have not experienced and therefore would not be expected to elicit a significant response during titration) and a relevant antigen presented in the context of HLA-DQ (in-groove peptide) are measured using ELISA. 96-well microtiter plates (Thermo Scientific) are coated overnight with tagged pMHC complexes containing the relevant peptide-in-groove or irrelevant antigen presented in the context of HLA-DQ in phosphate-buffered saline (PBS, Irvine Scientific). Plates are 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.

[0165] Pre- and post-immune antisera 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 the substrate according to the manufacturer's recommended procedure. The absorbance at 450 nm was recorded using a spectrophotometer (Victor, Perkin Elmer). Antibody titers were calculated using Graphpad PRISM software. The antibody titer was calculated as the interpolated serum dilution factor at which the binding signal was twice the background.

[0166] Tolerizing mice to human HLA class II molecules or portions thereof improves the ability of the mice to generate specific antibody responses against the pMHC of interest compared to control mice that have not been tolerized to human HLA class II molecules.

[0167] Example 4. Testing of various peptides in peptide-MHC II protein constructs Various soluble peptide-MHC II constructs with variations of the gliadin immunogen were designed to test various parameters of the MHC ligand peptides and to confirm expression of the constructs with the various ligand peptides. Specifically, variations of αI gliadin, αII gliadin, and ω2 gliadin (Table 6) were tested. [Table 6]

[0168] The portion of the full-length HLA-II DQα1 chain included in the soluble HLA-DQ2 constructs tested below included 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 included in the soluble HLA-DQ2 constructs tested below included residues 33-230 of SEQ ID NO:50 (SEQ ID NO:60). A description of the soluble peptide-MHC II constructs is shown in Table 7. Some constructs contained PADRE, although other T cell epitopes can also be used. As shown in Table 7, good yields were observed with all constructs. [Table 7]

[0169] Proteins are purified using standard procedures, including affinity and size-exclusion chromatography. The final amount of protein obtained after purification is determined by UV absorbance and the extinction coefficient calculated based on the amino acid composition of the protein. Production yield is calculated by dividing the mass of purified protein by the volume of culture medium.

[0170] Peptide-MHC constructs are used to test for their ability to bind to antibodies against MHC class II proteins via two different Biacore assays. In both assay formats, the instrument used is an Octet HTX, the chip type is an Octet biosensor coated with anti-mouse or anti-human Fc, the assay is run at 25°C, the running buffer is HBS-ET + 1 mg / mL BSA, the capture mixing speed and time are 1000 rpm and 1 minute, and the sample injection mixing speed and time are 1000 rpm and 2 minutes.

[0171] Each construct was analyzed to verify binding to the monoclonal antibodies. In the first experiment, approximately 0.8 nm of a pan-class II anti-HLA mAb or anti-DR / DQ mAb was captured by immersing an anti-mFc-coated octet biosensor in a well containing 100 nM of mAb for 1 minute. The mAb-captured sensor was then submerged in a well containing 200 nM of peptide-MHC constructs. The soluble peptide-MHC constructs bound to both anti-class II monoclonal antibodies captured on the anti-mFc sensor surface but not to the isotype control mAb. In the second experiment, approximately 1 nm of peptide-MHC constructs was captured by immersing an anti-hFc-coated octet biosensor in a well containing 200 nM of peptide-MHC constructs for 1 minute. The peptide-MHC-construct-captured sensor was then submerged in a well containing 100 nM of pan-class II anti-HLA mAb or anti-DR / DQ mAb. Soluble peptide-MHC constructs captured on the anti-hFc sensor surface bind to both anti-class II monoclonal antibodies but not to an isotype control mAb. The pan-class II anti-HLA antibody binds only to properly folded HLA proteins, verifying the conformational integrity of the produced and purified proteins.

[0172] Next, mice tolerized to empty MHC class II molecules are generated or provided, but the MHC class II molecules are not derived from a mouse (e.g., but not limited to, a human). For example, a first mouse expressing an MHC class II molecule derived from a corresponding endogenous locus or a locus other than the corresponding endogenous locus (e.g., but not limited to, the ROSA26 locus) is tolerized to the empty MHC class II molecule. These tolerized mice are then injected with an immunogen (e.g., an MHC class II molecule containing an immunogenic peptide in its groove, such as one of the peptide-MHC constructs in Example 4). These immunized mice produce specific antibody titers against this specific immunogen compared to mice that cannot tolerate the empty MHC class II molecule. Alternatively, mice that have not been tolerized and immunized with the target MHC class II molecule will produce antibodies that not only recognize the immunogenic peptide but also the MHC class II molecule. Thus, tolerized mice immunized with the MHC class II molecules described herein are capable of generating an antigen-specific immune response without generating antibodies directed solely against the MHC class II molecules.

[0173] Peptides as described in Example 4 for linking to HLA-DQB chains as DNA and soluble dimeric proteins are selected for immunization and screening.

[0174] Mice (e.g., mice containing humanized immunoglobulin heavy and / or light chain variable region loci) are immunized with a peptide-MHC (pMHC) complex of interest comprising a peptide antigenic to the mouse and a human or humanized MHC class II molecule to which the mouse is to be tolerized. The mice are additionally and optionally boosted with a pMHC complex of interest, which booster is optionally linked to a helper T cell epitope. Antibodies (e.g., human or humanized antibodies expressed from the humanized immunoglobulin heavy and / or light chain loci) are isolated from the immunized mice and tested for binding specificity to the pMHC complex.

[0175] Test mice are provided that are tolerized to human MHC II molecules and comprise nucleotide sequences encoding a humanized immunoglobulin heavy chain locus (see, e.g., Macdonald (2014) Proc. Natl. Acad. Sci. USA 111:5147-5152, which is incorporated by reference in its entirety for all purposes), and a humanized common light chain locus (see, e.g., U.S. Pat. Nos. 10,143,186, 10,130,081, and 9,969,814; U.S. Patent Application Publication Nos. 2012 / 0021409, 2012 / 0192300, 2013 / 0045492, 2013 / 0185821, 2013 / 0302836, and 2015 / 0313193, each of which is incorporated by reference in its entirety for all purposes). These test mice, as well as non-tolerized control mice containing a functional (e.g., murine) ADAM6 gene (see, e.g., U.S. Patent Nos. 8,642,835 and 8,697,940, each of which is incorporated by reference in its entirety for all purposes) and humanized immunoglobulin heavy and light chain loci, are immunized with pMHC complexes containing heterologous peptide inclusions presented in the context of HLA-DQ molecules, either administered as protein immunogens or DNA encoding the pMHC complexes. Mice are boosted at various time intervals via various routes using pMHC complex immunogens containing standard adjuvants or pMHC complex immunogens linked to a T helper pan-DR epitope (PADRE) peptide. Pre-immune serum is collected from the mice before the start of immunization. Mice are bled periodically, and antiserum titers are analyzed for each antigen.

[0176] Antibody titers in serum against an irrelevant antigen (i.e., an antigen that the mice have not experienced and therefore would not be expected to elicit a significant response during titration) and a relevant antigen presented in the context of HLA-DQ (in-groove peptide) are measured using ELISA. 96-well microtiter plates (Thermo Scientific) are coated overnight with tagged pMHC complexes containing the relevant peptide-in-groove or irrelevant antigen presented in the context of HLA-DQ in phosphate-buffered saline (PBS, Irvine Scientific). Plates are 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.

[0177] Pre- and post-immune antisera 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 the substrate according to the manufacturer's recommended procedure. The absorbance at 450 nm was recorded using a spectrophotometer (Victor, Perkin Elmer). Antibody titers were calculated using Graphpad PRISM software. The antibody titer was calculated as the interpolated serum dilution factor at which the binding signal was twice the background.

[0178] Tolerizing mice to human HLA class II molecules or portions thereof improves the ability of the mice to generate specific antibody responses against the pMHC of interest compared to control mice that have not been tolerized to human HLA class II molecules. The present invention provides, for example, the following items. (Item 1) 1. A composition comprising an MHC ligand peptide covalently linked to an 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, the MHC ligand peptide is covalently linked to the MHC class II molecule by a peptide linker; the MHC ligand peptide or the peptide linker comprises a first cysteine ​​and the MHC class II molecule comprises a second cysteine; The composition, wherein the first cysteine ​​and the second cysteine ​​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 portion thereof and the MHC class II β chain or portion thereof. (Item 2) 2. The composition of claim 1, wherein the MHC class II α chain or the portion thereof comprises an α1 domain and the MHC class II β chain or the portion thereof comprises a β1 domain. (Item 3) 3. The composition of claim 2, wherein the MHC class II α chain or the portion thereof comprises an MHC class II α chain extracellular domain, and the MHC class II β chain or the portion thereof comprises an MHC class II β chain extracellular domain. (Item 4) (1) the MHC class II α chain or the portion thereof comprises the α1 domain, the α2 domain, the transmembrane domain, and the cytoplasmic domain; (2) The composition according to item 2 or 3, wherein the MHC class II β chain or the portion thereof comprises the β1 domain, the β2 domain, the transmembrane domain, and the cytoplasmic domain. (Item 5) Item 10. The composition of any one of the preceding items, wherein the composition is immobilized on a membrane. (Item 6) 4. The composition according to any one of items 1 to 3, wherein the composition is soluble. (Item 7) (1) the MHC class II α chain or the portion thereof comprises the α1 domain and the α2 domain, but does not comprise a transmembrane domain or a cytoplasmic domain; (2) The composition according to item 6, wherein the MHC class II β chain or the portion thereof comprises the β1 domain and the β2 domain but does not comprise a transmembrane domain or a cytoplasmic domain. (Item 8) 8. The composition of claim 6 or 7, wherein the MHC class II α chain or portion thereof and the MHC class II β chain or portion thereof are linked by a Jun-Fos zipper, electrostatic engineering, knobs-into-holes, an immunoglobulin scaffold, an immunoglobulin Fc region, or a linker. (Item 9) If the MHC class II α chain or the part thereof and the MHC class II β chain or a portion thereof is linked by a Jun-Fos zipper containing a Jun leucine zipper dimerization motif and a Fos leucine zipper dimerization motif, 9. The composition of claim 8, wherein the MHC class II α chain or portion thereof is linked to the Jun leucine zipper dimerization motif and the MHC class II β chain or portion thereof is linked to the Fos leucine zipper dimerization motif, or the MHC class II α chain or portion thereof is linked to the Fos leucine zipper dimerization motif and the MHC class II β chain or portion thereof is linked to the Jun leucine zipper dimerization motif. (Item 10) the C-terminus of the MHC class II α chain or portion thereof is linked to the Jun leucine zipper dimerization motif and the C-terminus of the MHC class II β chain or portion thereof is linked to the Fos leucine zipper dimerization motif; or 10. The composition of claim 9, wherein the C-terminus of the MHC class II α chain or portion thereof is linked to the Fos leucine zipper dimerization motif and the C-terminus of the MHC class II β chain or portion thereof is linked to the Jun leucine zipper dimerization motif. (Item 11) the MHC class II α chain or portion thereof is linked to the Jun leucine zipper dimerization motif by an MHC-Jun linker, and the MHC class II β chain or portion thereof is linked to the Fos leucine zipper dimerization motif by an MHC-Fos linker; or 11. The composition of claim 9 or 10, wherein the MHC class II α chain or the portion thereof is linked to the Fos leucine zipper dimerization motif by the MHC-Fos linker, and the MHC class II β chain or the portion thereof is linked to the Jun leucine zipper dimerization motif by the MHC-Jun linker. (Item 12) Item 12. The composition of item 11, wherein the MHC-Jun linker and the MHC-Fos linker each comprise the sequence set forth in SEQ ID NO: 1. (Item 13) 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; or Item 11. The composition of any one of the preceding items, wherein the MHC ligand peptide comprises residues P-1 to P9 or residues P-3 to P9. (Item 14) Item 11. The composition of any one of the preceding items, wherein the MHC ligand peptide is an antigenic MHC ligand peptide. (Item 15) Item 11. The composition of any one of the preceding items, wherein the MHC ligand peptide is associated with a T cell mediated disease. (Item 16) 10. The composition of any one of the preceding items, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule is a flexible linker. (Item 17) 10. The composition of any one of the preceding claims, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule comprises one or more flexible amino acids and one or more polar amino acids. (Item 18) 10. The composition of any one of the preceding items, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule does not contain any charged amino acids. (Item 19) 10. The composition of any one of the preceding items, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule comprises a cleavage site. (Item 20) 20. The composition of claim 19, wherein the cleavage site is a tobacco etch virus (TEV) protease cleavage site. (Item 21) Item 11. The composition of any one of the preceding items, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule is non-immunogenic. (Item 22) 2. The composition of any one of the preceding claims, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule is attached to the N-terminus of the MHC class II β chain or portion thereof. (Item 23) 2. The composition of any one of the preceding claims, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule is attached to the N-terminus of the MHC class II alpha chain or portion thereof. (Item 24) Item 11. The composition of any one of the preceding items, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule is at least about 9 amino acids in length. (Item 25) 2. The composition of any one of the preceding items, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule is about 9 to about 50 amino acids in length. (Item 26) 10. The composition of any one of the preceding items, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule comprises 2 to 4 repeats of the sequence set forth in SEQ ID NO:4. (Item 27) 10. The composition of any one of the preceding items, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule comprises the first cysteine. (Item 28) 28. The composition of claim 27, wherein the first cysteine ​​is the only cysteine ​​in the peptide linker linking the MHC ligand peptide to the MHC class II molecule. (Item 29) 29. The composition of claim 27 or 28, wherein the first cysteine ​​is in the first four amino acids of the peptide linker that links the MHC ligand peptide to the MHC class II molecule. (Item 30) 10. The composition of claim 1, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule comprises two to four repeats of the sequence set forth in SEQ ID NO: 4, and one amino acid in one of the repeats is mutated to cysteine. (Item 31) 31. The composition of claim 30, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule comprises the sequence set forth in SEQ ID NO: 21. (Item 32) 27. The composition of any one of items 1 to 26, wherein the MHC ligand peptide comprises the first cysteine. (Item 33) 33. The composition of claim 32, wherein the first cysteine ​​faces away from the epitope formed by the composition. (Item 34) Item 11. The composition of any one of the preceding items, wherein the second cysteine ​​is in the MHC class II α chain or portion thereof. (Item 35) 35. The composition of claim 34, wherein the peptide linker linking the MHC ligand peptide to the MHC class II molecule is attached to the N-terminus of the MHC class II β chain or portion thereof. (Item 36) Item 11. The composition of any one of the preceding items, wherein the second cysteine ​​is not present in a wild-type MHC class II molecule corresponding to the MHC class II molecule in the composition. (Item 37) 37. The composition of claim 36, wherein the second cysteine ​​is present in place of a non-cysteine ​​amino acid in the corresponding wild-type MHC class II molecule. (Item 38) the second cysteine ​​is in the MHC class II α chain or portion thereof; 38. The composition of claim 37, wherein the second cysteine ​​is at a position corresponding to position 101 of the sequence set forth in SEQ ID NO: 49 when the MHC class II α chain or the portion thereof is optimally aligned with SEQ ID NO: 49. (Item 39) 2. The composition of any one of the preceding items, wherein the MHC class II molecule lacks a cysteine ​​present in the corresponding wild-type MHC class II molecule. (Item 40) 40. The composition of claim 39, wherein the cysteine ​​present in the corresponding wild-type MHC class II molecule is substituted with alanine or glutamine in the MHC class II molecule in the composition. (Item 41) 39. The composition of any one of items 1 to 38, wherein the MHC class II α chain or the portion thereof lacks a cysteine ​​present in the corresponding wild-type MHC class II α chain. (Item 42) 42. The composition of claim 41, wherein the cysteine ​​present in the corresponding wild-type MHC class II α chain is substituted with alanine or glutamine in the MHC class II α chain or portion thereof in the composition. (Item 43) 43. The composition of claim 41 or 42, wherein the cysteine ​​in the corresponding wild-type MHC class II α chain is at a position corresponding to position 70 of the sequence set forth in SEQ ID NO: 49 when the MHC class II α chain or the portion thereof is optimally aligned with SEQ ID NO: 49. (Item 44) The composition of any one of the preceding items, wherein the composition further comprises one or more immunostimulatory molecules. (Item 45) 45. The composition of claim 44, wherein the one or more immunostimulatory molecules comprise a pan-DR-binding epitope (PADRE) and / or a peptide derived from lymphocytic choriomeningitis virus (LCMV). (Item 46) 46. ​​The composition of claim 44 or 45, wherein the one or more immunostimulatory molecules are directly or indirectly covalently linked to the MHC class II molecule. (Item 47) 47. The composition of any one of items 44 to 46, wherein the one or more immunostimulatory molecules are directly or indirectly covalently bound to the MHC class II α chain or portion thereof and / or the MHC class II β chain or portion thereof. (Item 48) Item 10. The composition of any one of the preceding items, wherein the MHC class II molecule is a human MHC class II molecule. (Item 49) 49. The composition of claim 48, wherein the human MHC class II molecule is selected from the group consisting of HLA-DQ, HLA-DR, and HLA-DP. (Item 50) 50. The composition of claim 49, wherein the human MHC class II molecule is an HLA-DQ2 molecule. (Item 51) 50. The composition of claim 49, wherein the human MHC class II molecule is an HLA-DR2 molecule. (Item 52) the MHC class II α chain or the portion thereof comprises an MHC class II α chain extracellular domain, and the MHC class II β chain or the portion thereof comprises an MHC class II β chain extracellular domain; the peptide linker linking the MHC ligand peptide to the MHC class II molecule is a flexible linker having a length of about 9 to about 50 amino acids and containing a first cysteine, and is connected to the N-terminus of the MHC class II β chain or the portion thereof; the second cysteine ​​is in the MHC class II α chain or portion thereof and is not present in a wild-type MHC class II molecule corresponding to the MHC class II molecule in the composition; 2. The composition of any one of the preceding items, wherein the MHC class II molecule lacks a cysteine ​​present in the corresponding wild-type MHC class II molecule. (Item 53) the composition is soluble; the MHC class II α chain or the portion thereof comprises the α1 and α2 domains but does not comprise a transmembrane or cytoplasmic domain; the MHC class II β chain or the portion thereof comprises the β1 domain and the β2 domain, but does not comprise a transmembrane domain or a cytoplasmic domain; 53. The composition of claim 52, wherein the MHC class II α chain or portion thereof and the MHC class II β chain or portion thereof are linked by a Jun-Fos zipper comprising a Jun leucine zipper dimerization motif and a Fos leucine zipper dimerization motif. (Item 54) the second cysteine ​​is at a position corresponding to position 101 of the sequence set forth in SEQ ID NO:49 when the MHC class II α chain or portion thereof is optimally aligned with SEQ ID NO:49; 54. The composition of claim 52 or 53, wherein the cysteine ​​in the corresponding wild-type MHC class II molecule is at a position corresponding to position 70 of the sequence set forth in SEQ ID NO: 49 when the MHC class II α chain or the portion thereof is optimally aligned with SEQ ID NO: 49. (Item 55) 55. The composition of any one of items 52 to 54, wherein 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. (Item 56) 56. The composition of item 55, wherein the human MHC class II molecule is HLA-DQ. (Item 57) A nucleic acid encoding the composition of any one of the preceding items. (Item 58) 57. A method for inducing an immune response in a subject, the method comprising administering to the subject an effective amount of the composition of any one of items 1 to 56 or a nucleic acid encoding said composition. (Item 59) 1. A method for producing an antigen binding protein that specifically binds to an antigenic composition comprising an MHC ligand peptide covalently linked to an MHC class II molecule, comprising: (a) immunizing a non-human animal with the composition according to any one of items 1 to 56 or a nucleic acid encoding said composition; (b) maintaining the non-human animal in conditions sufficient for the non-human animal to mount an immune response to the composition. (Item 60) 1. A method of producing an antigen binding protein, comprising: (a) immunizing a non-human animal with the composition according to any one of items 1 to 56 or a nucleic acid encoding said composition; (b) maintaining the non-human animal in conditions sufficient for the non-human animal to mount an immune response to the composition. (Item 61) 61. The method of claim 60, wherein the antigen binding protein specifically binds to an antigenic composition comprising an MHC ligand peptide covalently linked to an MHC class II molecule.

Claims

[Claim 1] The invention described in the specification.

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