MHC class ii molecules and methods of using the same

Abstract

The present disclosure relates to HLA class II molecules having higher affinity for CD4 than naturally occurring HLA class II molecules. In certain aspects, the HLA class II molecules comprise a DR beta chain having: (i) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (ii) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (iii) or both (i) and (ii). Certain aspects of the present disclosure relate to nucleic acid molecules encoding the HLA class II molecules, vectors comprising the nucleic acid molecules, cells comprising them, and methods of using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims the benefit of priority to U.S. Provisional Application No. 62 / 880,509, filed on July 30, 2019, and U.S. Provisional Application No. 63 / 029,115, filed on May 22, 2020, each of which is incorporated herein by reference in its entirety.

[0003] Citations of sequence listings submitted electronically via EFS-WEB

[0004] The contents of this electronically submitted Sequence Listing (Name: 4285-012PC02_SL_ST25.txt, Size: 63,907 bytes; Creation Date: July 28, 2020) are incorporated herein by reference in their entirety. Field of the Invention

[0005] The present disclosure provides major histocompatibility complex (MHC) class II molecules with increased affinity for CD4 and uses thereof. Background of the Invention

[0007] Immunotherapy has become a key tool in the fight against a variety of diseases, including cancer. T cell therapy is at the forefront of immunotherapy development, and adoptive transfer of anti-tumor T cells has been shown to induce clinical responses in cancer patients.

[0008] Directed T cell therapy using T cell receptors (TCRs) expressed by T cells that are specific for target epitopes expressed by tumor cells is a promising form of T cell therapy. Antigen presenting cells display peptide fragments associated with the major histocompatibility complex (MHC) on their surface to induce an immune response. Improved presentation of endogenous peptides by class II has been shown to be associated with improved survival in cancer patients. However, the development of new TCRs that can specifically target MHC class II-presented peptides has been hampered by the low affinity of MHC class II proteins for CD4 expressed by T cells.

[0009] The present disclosure provides MHC class II proteins with increased affinity for CD4 and methods of using them to identify and develop new MHC class II-specific TCRs. Summary of the Invention

[0010] Certain aspects of the present disclosure relate to HLA class II molecules comprising a DR β chain, wherein the DR β chain comprises an amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 1.

[0011] Certain aspects of the present disclosure relate to an HLA class II molecule comprising a DR beta chain, wherein the DR beta chain comprises a substitution mutation at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, wherein the substitution mutation is a substitution with an amino acid other than leucine.

[0012] In certain embodiments, the DR beta chain further comprises an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1.

[0013] Certain aspects of the present disclosure relate to an HLA class II molecule comprising a DR beta chain, wherein the DR beta chain comprises an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1.

[0014] Certain aspects of the present disclosure relate to an HLA class II molecule comprising a DR beta chain, wherein the DR beta chain comprises a substitution mutation at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, wherein the substitution mutation is a substitution with an amino acid other than valine.

[0015] In certain embodiments, the DR beta chain further comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1. In certain embodiments, the DR beta chain further comprises an amino acid other than asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 1. In certain embodiments, the DR beta chain further comprises an amino acid other than isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 1. In certain embodiments, the DR beta chain further comprises an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1. In certain embodiments, the DR beta chain further comprises an amino acid other than proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 1.

[0016] In certain embodiments, the beta chain of the HLA class II molecule comprises a DR2, DR3, DR4, DR5, DR6, DR7, DR8, DR9, DR10, DR11, DR12, DR13, DR14, DR15, or DR16 allele. In certain embodiments, the beta chain of the MHC class II molecule comprises an HLA allele selected from the group consisting of DRB1*01, DRB1*03, DRB1*04, DRB1*07, DRB1*08, DRB1*09, DRB1*10, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*15, and DRB1*16.

[0017] In certain embodiments, the HLA class II molecule further comprises an alpha chain. In certain embodiments, the alpha chain of the MHC class II molecule comprises an HLA-DRA1*01 allele.

[0018] In certain embodiments, the beta chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1; (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1; and (c) at least two of the following: (i) an amino acid other than leucine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1; (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1; (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1; (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1; and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0019] In certain embodiments, the beta chain comprises (c) at least three of the following: (i) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1; (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1; (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1; (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1; and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0020] In certain embodiments, the beta chain comprises (c) at least four of: (i) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1; (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1; (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1; (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1; and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0021] In certain embodiments, the beta chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, and (d) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1.

[0022] In certain embodiments, the beta chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (d) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1, (e) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1, (f) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1, (g) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1, and (h) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0023] In certain embodiments, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 1 comprises a hydrophobic side chain. In certain embodiments, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 1 is selected from the group consisting of alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain embodiments, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 1 is tryptophan.

[0024] In certain embodiments, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 1 comprises a hydrophobic side chain. In certain embodiments, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 1 is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain embodiments, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 1 is methionine.

[0025] In certain embodiments, the beta chain of the MHC class II molecule comprises an amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 1. In certain embodiments, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 1 is selected from arginine, histidine, and lysine. In certain embodiments, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 1 is histidine.

[0026] In certain embodiments, the beta chain of the MHC class II molecule comprises an amino acid other than lysine at the position corresponding to amino acid residue 139 of SEQ ID NO: 1. In certain embodiments, the amino acid other than lysine at the position corresponding to amino acid residue 139 of SEQ ID NO: 1 is selected from serine, threonine, and glutamine. In certain embodiments, the amino acid other than lysine at the position corresponding to amino acid residue 139 of SEQ ID NO: 1 is threonine.

[0027] In certain embodiments, the beta chain of the MHC class II molecule comprises an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1. In certain embodiments, the amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1 is selected from the group consisting of serine, asparagine, threonine, and glutamine. In certain embodiments, the amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1 is glutamine.

[0028] In certain embodiments, the beta chain of the MHC class II molecule comprises an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1. In certain embodiments, the amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1 is selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain embodiments, the amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1 is isoleucine.

[0029] In certain embodiments, the beta chain of the MHC class II molecule comprises an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1. In certain embodiments, the amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1 is selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain embodiments, the amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1 is methionine.

[0030] In certain embodiments, the beta chain of the MHC class II molecule comprises an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1. In certain embodiments, the amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1 is selected from the group consisting of serine, asparagine, threonine, and glutamine. In certain embodiments, the amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1 is threonine.

[0031] In certain embodiments, the beta chain comprises: (a) tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (b) methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (c) histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, and (d) isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1.

[0032] In certain embodiments, the DR beta chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 5.

[0033] In certain embodiments, the beta chain comprises (a) tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 1; (b) methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1; and (c) at least two of the following: (i) histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (ii) threonine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1, (iii) glutamine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1, (iv) isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1, (v) methionine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1, and (vi) threonine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0034] In certain embodiments, the beta chain comprises (a) a tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 1; (b) a methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1; and (c) at least three of: (i) a histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (ii) a threonine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1, (iii) a glutamine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1, (iv) an isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1, (v) a methionine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1, and (vi) a threonine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0035] In certain embodiments, the beta chain comprises (a) a tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 1; (b) a methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1; and (c) at least four of: (i) a histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (ii) a threonine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1, (iii) a glutamine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1, (iv) an isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1, (v) a methionine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1, and (vi) a threonine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0036] In certain embodiments, the beta chain comprises (a) tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (b) methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (c) histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (d) threonine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1, (e) glutamine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1, (f) isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1, (g) methionine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1, and (h) threonine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0037] In certain embodiments, the DR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the DR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0038] In certain embodiments, the beta chain of the HLA class II molecule comprises a DR1, DR3, DR4, DR7, DR8, DR9, DR10, DR11, DR12, DR13, DR14, DR15, or DR16 allele. In certain embodiments, the beta chain of the MHC class II molecule comprises an HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*06, HLA-DRB1*07, HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*10, HLA-DRB1*11, HLA-DRB1*12, HLA-DRB1*13, HLA-DRB1*14, HLA-DRB1*15, or HLA-DRB1*16 allele. In certain embodiments, the alpha chain of the MHC class II molecule comprises an HLA-DRA1*01 allele.

[0039] In certain embodiments, the DR alpha chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 6 or 8. In certain embodiments, the DR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 6 or 8.

[0040] In certain embodiments, the DR beta chain has increased affinity for a CD4 protein compared to a reference HLA class II molecule, wherein the DR beta chain comprised by the reference HLA class II molecule comprises (i) a leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 and / or (ii) a valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1.

[0041] In certain embodiments, the increased affinity is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold.

[0042] In certain embodiments, the DR beta chain is associated with the membrane of a cell. In certain embodiments, the DR beta chain is not associated with the membrane of a cell. In certain embodiments, the DR beta chain comprises the extracellular domain of a full-length DR beta chain. In certain embodiments, the DR beta chain does not comprise the transmembrane domain of a full-length DR beta chain.

[0043] In certain embodiments, the DR alpha chain is associated with the membrane of a cell. In certain embodiments, the DR alpha chain is not associated with the membrane of a cell. In certain embodiments, the DR alpha chain comprises the extracellular domain of a full-length DR alpha chain. In certain embodiments, the DR alpha chain does not comprise the transmembrane domain of a full-length DR alpha chain.

[0044] In certain embodiments, the DR beta chain is attached to or associated with an inert particle. In certain embodiments, the inert particle is a bead. In certain embodiments, the inert particle is a nanoparticle. In certain embodiments, the nanoparticle is selected from the group consisting of polyethylene glycolated iron oxide, chitosan, dextran, gelatin, alginate, liposome, starch, branched polymer, carbon-based carrier, polylactic acid, poly(cyano)acrylate, polyethylenimine, block copolymer, polycaprolactone, SPIONS, USPIONS, Cd / Zn-selenide, or silica nanoparticle. In certain embodiments, the nanoparticle is a polyethylene glycolated iron oxide nanoparticle.

[0045] In certain embodiments, the DR beta chain comprises a signal peptide. In certain embodiments, the DR alpha chain comprises a signal peptide. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0046] Certain aspects of the present disclosure relate to nucleic acid molecules encoding a DR beta chain disclosed herein. In certain embodiments, the nucleic acid molecule further encodes a DR alpha chain disclosed herein. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 2.

[0047] Certain aspects of the present disclosure relate to vectors comprising a nucleic acid molecule disclosed herein.

[0048] Certain aspects of the present disclosure relate to cells comprising an HLA class II molecule disclosed herein, a nucleic acid molecule disclosed herein, or a vector disclosed herein. In certain embodiments, the cell is a mammalian cell or an insect cell. In certain embodiments, the cell is selected from the group consisting of a K562 cell, T2, HEK293, HEK293T, A375, SK-MEL-28, Me275, COS, fibroblast, tumor cell, or any combination thereof. In certain embodiments, the cell lacks endogenous MHC class II DR beta chain expression. In certain embodiments, the cell lacks endogenous MHC class II DR alpha chain expression.

[0049] Certain aspects of the present disclosure relate to a method of identifying a T cell receptor capable of binding an epitope in an MHC class II complex, the method comprising pulsing a cell disclosed herein with one or more peptides comprising the epitope, and stimulating one or more CD4 + T cells.

[0050] Certain aspects of the present disclosure relate to a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an MHC class II molecule disclosed herein. In certain embodiments, the disease or condition is a cancer or an infection.

[0051] In certain embodiments, the cancer is selected from the group consisting of melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, uterine cancer, lung cancer, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), esophageal cancer, small bowel cancer, urethral cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), bladder cancer, kidney or ureter cancer, renal pelvis cancer, glioma, squamous cell carcinoma, and combinations of said cancers.

[0052] In certain embodiments, the cancer is relapsed or refractory. In certain embodiments, the cancer is locally advanced. In certain embodiments, the cancer is advanced. In certain embodiments, the cancer is metastatic.

[0053] In some aspects, the HLA class II molecule binds CD4 with a Kd of less than about 100 µM. D In some aspects, the HLA class II molecule binds CD4 with a Kd of less than about 20 µM. D In some aspects, the HLA class II molecule binds CD4 with a Kd of less than about 20 µM. D In some aspects, the HLA class II molecule binds CD4 with a Kd of less than about 20 µM.

[0054] Certain aspects of the present disclosure relate to complexes comprising an HLA class II molecule disclosed herein and a peptide, wherein the peptide is selected from NY-ESO1 91-110 , Bet V 1 142-153 , HIV Gag 293-312 , HA 306-318 , Flu-HA 5-24 , Flu-HA 117-136 , Flu-HA 232-251 , Flu-HA 268-287 , Flu-HA 306-318 , HSD17B12 225-244 , LY6K 99-118 , and any combination thereof. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figures 1A-1F Data is provided illustrating the enhanced CD4 binding capacity of the modified DR molecules. Figure 1A is a table comparing the amino acid sequences of DPB1*04:01, DRB1*01:01 and DRB1*01:01 L114W / V143M+6reps , where mutated amino acids are underlined. Figure 1B and Figure 1C is a graphical representation of data from class II deficient K562 cells that were transduced with wild type DR1 (DRA1*01:01 / DRB1*01:01), DR1 L114W / V143M , DR1 L114W / V143M+6reps , wild type DP4 or DP4 L112W / V141M transduced and stained with sCD4. Figures 1D-1E shows DR1 L114W / V143M+6reps mutants (stained similarly with sCD4) Figure 1D ) and DRB1 L114W / V143M+2reps ( Figure 1E) were used to investigate the CD4 binding ability of a series of K562 derivatives. Figure 1F is a table listing the amino acid sequence of DPB1*04:01 and comparing the DRB1 alleles of DR3, DR4, DR7, DR10, DR11, and DR13 to DRB1 L114W / V143M+6reps and DRB1 L114W / V143M+2reps The alleles are compared together, with the mutated amino acid underlined. Figures 1G-1L This is a graphical representation of data showing that the L114W / V143M + 2 reps mutation enhances DR3, DR4, DR7, DR10, DR11, and DR13 binding to CD4 better than the L114W / V143M + 6 reps mutation. At least two independent experiments were performed. *, P < 0.05, by Student's t-test. Bars and error bars represent the mean ± SD of triplicate experiments. Figures 1M-1N Figure 2 is a biolayer interferometry sensorgram showing the interaction of biotinylated HLA-DR1 (ligand) with soluble CD4 (analyte) over a range of concentrations. Figure 1M ) and DR1 L114W / V143M+2reps ( Figure 1N ) were performed in parallel, and the binding experiments of wild-type DR1 ( Figure 1M ) No binding was detected. Figure 1O Figure 2 shows DR1 quantified by steady-state analysis. L114W / V143M+2reps Figure 3. Affinity between α and CD4. All data are representative of two independent experiments.

[0056] Figures 2A-2D Figure 1 is a graphical representation of affinity-matured DR dimers detected by homologous TCRs expressed in human primary CD4+ T cells. Figure 2A ), DR7-restricted TCR (SD334) ( Figure 2B ) and DR11-restricted TCR (F24) ( Figure 2C ) were reconstituted in primary human T cells and by the corresponding DR L114W / V143M+2reps Dimer staining. L114W / V143M+2reps Dimer and anti-Vβ22 mAb staining of DR11-restricted F24-transduced CD4 + T cells ( Figure 2D ). Note that F24 represents Vβ22. At least two independent experiments were performed.

[0057] Figures 3A-3D HLA-DR1 L114W / V143M+2reps Figure 3 shows the model structure of the complex with human CD4. Figure 3AAn overview ribbon model of the ternary complex structure of DRA1*01:01, DRB1*01:01, and CD4 is provided. Figures 3B-3D Provide wild-type DR1 (left) and mutant DR1 L114W / V143M+2reps (right) Close-up view of the following four mutated residues: L114W and V143M ( Figure 3B )、S118H( Figure 3C ) and T157I( Figure 3D ), as illustrated using a bat and stick.

[0058] Figures 4A-4II This is a graphical representation of a histogram illustrating the relative expression levels of HLA class II genes. HLA-DR and its derivatives were reconstituted in K562 cells and stained with anti-HLA class II monoclonal antibodies. Surface expression of all DR alleles was detected using anti-HLA class II monoclonal antibody clone 9-49 (I3). The open histogram represents isotype control staining.

[0059] Figures 5A-5L Figures 5A-5L DR1 is used to display the experience L114W / V143M+2reps In vitro staining of influenza virus hemagglutinin-specific peripheral CD4 dimers + Graphical representation of T cells. From two DR1 + Donor (No. 07 ( Figures 5A-5L Figure 5F) and No. 08 (Figure 5G to Figure 5L Purification of memory CD4 + T cells, and without in vitro stimulation, were treated with Flu-HA 5-24 (Figure 5B and Figure 5H), Flu-HA 117-136 (Figure 5C and Figure 5I), Flu-HA 232-251 (Figure 5D and Figure 5J), Flu-HA 268-287 (Figure 5E and Figure 5K) and Flu-HA 306-318 (Figure 5F and Figures 6A-6X ) Influenza virus hemagglutinin (Flu-HA) peptide has specific DR1 L114W / V143M+2reps Dimer staining. CLIP peptide was used as a negative control (Figure 5A and Figure 5G).

[0060] Figures 6A-6X Figures 6A-6X Yes Display DR1 L114W / V143M+6reps and DR1 L114W / V143M+2reps Robust dimer staining of HA1.7-transduced CD4 + Graphical representation of T cells. +Reconstitution in T cells was followed by induction with wild-type DR1 (Fig. 6I and 6M), DR1 in the absence of transduced TCR (Fig. 6I to Fig. 6L) and with transduction with HA1.7 TCR (Fig. 6M to Fig. 6P). L114W / V143M (Figure 6J and Figure 6N), DR1 L114W / V143M+6reps (Figures 6K and 6O) and DR1 L114W / V143M+2reps (Fig. 6L and 6P) Dimer staining, using CLIP dimer as a negative control ( Figures 6A-6X Figures 6A-6X ). In addition, HA1.7 was expressed in primary CD4 + T cells were reconstituted and then treated with Flu-HA 306-318 Specific DR1 L114W / V143M+2reps Dimer ( Figures 6A-6X Figures 7A-7O ) or wild-type DR1 dextramer ( Figures 7A-7O Figure 7D )dyeing.

[0061] Figures 7A-7D Figures 7A-7D is a graphical representation of data showing the use of affinity matured dimers to clone DR1-restricted TCRs. + Purified primary CD4 + T cells and irradiated HSD17B12 225-244 shock (Figure 7B) and LY6K 99-118 Impact Figures 7A-7D ) were stimulated with DR1-expressing aAPC. L114W / V143M+2reps Dimers stimulated by CD4 + T cells were stained ( Figures 7A-7D Figures 7A-7D ). DR1-restricted TCR was expressed in primary CD4 + T cells and stained with the corresponding dimers ( Figures 7A-7D Figures 4A-4II ). In the IL-2 ELISPOT assay, cells expressing DR1-restricted DR1-07-HSD17B12 225-244 ( Figure 1A ) and DR1-08-LY6K 99-118 ( Figure 1B )TCR primary CD4 + T cells were induced by HSD17B12 225-244 ( Figure 1C ) and LY6K 99-118 ( Figure 1D ) Peptide-pulsed DR1-K562 cells. DETAILED DESCRIPTION

[0062] The present disclosure relates to MHC class II molecules with increased affinity for CD4. In some aspects, the present disclosure relates to MHC class II molecules comprising an HLA-DR (DR) beta chain, wherein the DR beta chain has increased affinity for CD4.

[0063] The present disclosure further relates to MHC class II molecules comprising a DR beta chain, wherein the DR beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1. In some aspects, the DR beta chain further comprises an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1.

[0064] The present disclosure further relates to MHC class II molecules comprising a DR beta chain, wherein the DR beta chain comprises an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1. In some aspects, the DR beta chain further comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1.

[0065] In some aspects, the DR beta chain further comprises at least two of: (i) a histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (ii) a threonine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1, (iii) a glutamine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1, (iv) an isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1, (v) a methionine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1, and (vi) a threonine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0066] In some aspects, the DR beta chain comprises (a) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1; (b) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1; (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1; and (d) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1.

[0067] I. Terms

[0068] To enable the disclosure to be more readily understood, certain terms are first defined. As used in this application, unless specifically stated otherwise, each of the following terms has the meaning set forth below. Additional definitions are set forth throughout the application.

[0069] It should be noted that the terms "a" and "an" entity refer to one or more than one of that entity; for example, "a nucleotide sequence" should be understood as meaning one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0070] Also, as used herein, "and / or" shall, where used in this document, be understood to mean either each of the two specified features or components individually, or both of the specified features or components together with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to cover the aspects of "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0071] The term "about" is used herein to mean approximately, roughly, around, or in the vicinity of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values. Generally, the term "about" is used herein to modify a numerical value to within 10% above and below the value.

[0072] It should be appreciated that whenever aspects are described herein with the wording "comprising" various aspects, additional analogous aspects described with the wording "consisting of and / or "consisting essentially of are also provided.

[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, provide one of ordinary skill with a general dictionary of many of the terms used in this disclosure.

[0074] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxyl orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0075] “Administering” refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, e.g., by injection or infusion. As used herein, the phrase“parenteral administration” means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some aspects, the formulation is administered by a non-parenteral route, e.g., orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, e.g., intranasal, vaginal, rectal, sublingual, or topical. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0076] As used herein, the term“HLA” refers to human leukocyte antigen. In humans, HLA genes encode major histocompatibility complex (MHC) proteins. MHC proteins are expressed on the surface of cells and are involved in activation of immune responses. HLA class II genes encode MHC class II proteins that are expressed on the surface of professional antigen presenting cells (APCs). Non-limiting examples of professional APCs include monocytes, macrophages, dendritic cells (DCs), and B lymphocytes. Some endothelial cells and epithelial cells also express MHC class II molecules upon activation of inflammatory signals. Humans lacking functional MHC class II molecules are highly susceptible to a range of infectious diseases and typically die in early childhood.

[0077] As used herein,“HLA class II molecule” or“MHC class II molecule” refers to the protein product of a wild-type or variant HLA class II gene that encodes a MHC class II molecule. Thus,“HLA class II molecule” and“MHC class II molecule” are used interchangeably herein. A typical MHC class II molecule comprises two protein chains: an alpha chain and a beta chain. Generally, each of the naturally occurring alpha and beta chains comprises a transmembrane domain that anchors the alpha / beta chain to the surface of a cell, and an extracellular domain that carries an antigen and interacts with a TCR expressed on a T cell and / or CD4.

[0078] Both MHC class II alpha and beta chains are encoded by the HLA gene complex. The HLA complex is located within the 6p21.3 region of human chromosome 6 and comprises more than 220 genes with different functions. It is known in the art that the HLA gene complex is highly variable, with over 20,000 HLA alleles and related alleles, including over 250 MHC class II alpha chain alleles and 5,000 MHC class II beta chain alleles, encoding thousands of MHC class II proteins (see, e.g., hla.alleles.org, last accessed May 20, 2019, incorporated by reference herein in its entirety). For example, one such HLA-DP allele, DP4, is the most common allele in many ethnic populations. Each alpha chain and beta chain is typically expressed as a proprotein, which further comprises a signal peptide that is cleaved off. Any number of naturally occurring signal peptides can be used to facilitate expression and localization of the alpha and beta chains disclosed herein. One such example is SEQ ID NO: 9.

[0079] Three loci in the HLA complex encode MHC class II proteins: HLA-DP, HLA-DQ, and HLA-DR. HLA-DO and HLA-DM encode proteins that associate with MHC class II molecules and support their conformation and function. Representative HLA-DR sequences are provided in Table 1.

[0080] Table 1: DR beta chain and alpha chain amino acid and nucleotide sequences.

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] When MHC class II molecules are complexed with an antigenic peptide, an antigenic peptide 10-30 amino acids long binds the peptide binding groove and is presented extracellularly to CD4+ cells. Both the alpha and beta chains fold into two separate domains; alpha-1 and alpha-2 of the alpha polypeptide, and beta-1 and beta-2 of the beta polypeptide. The invariant residues at LI 14, VI 16, VI 43, LI 58, and M160 that are recognized and bound by CD4 are located in the beta-2 domain of the beta polypeptide. An open peptide binding groove that holds the antigen for presentation is found between the alpha-1 and beta-1 domains. In interaction with CD4+ T cells, MHC class II complexes interact with T cell receptors (TCRs) expressed on the surface of T cells. In addition, the beta chain of MHC class II molecules weakly interacts with CD4 expressed on the surface of T cells (K D >2mM). The canonical CD4 amino acid sequence (UniProt-P01730) is provided in Table 2 (SEQ ID NO: 10).

[0087] Table 2: Human CD4 amino acid sequence

[0088]

[0089] As used herein, the term “T cell receptor” (TCR) refers to a heteromeric cell surface receptor capable of specific interaction with a target antigen. As used herein, “TCR” includes, but is not limited to, naturally occurring and non-naturally occurring TCRs, full-length TCRs and antigen-binding portions thereof, chimeric TCRs, TCR fusion constructs, and synthetic TCRs. In humans, TCRs are expressed on the surface of T cells, and they are responsible for T cell recognition and targeting of antigen-presenting cells. Antigen-presenting cells (APCs) display fragments of foreign proteins (antigens) complexed with major histocompatibility complexes (MHC class I or MHC class II) (also referred to herein as complexed with HLA molecules, e.g., HLA class II molecules). TCRs recognize and bind to the peptide:HLA complex and recruit CD8 (for MHC class I molecules) or CD4 (for MHC class II molecules) expressed by the T cell, thereby activating the TCR. The activated TCR initiates downstream signaling and immune responses, including destruction of the APC.

[0090] Generally, a TCR can comprise two chains, an alpha chain and a beta chain (or, less commonly, a gamma chain and a delta chain), which are interconnected to each other by disulfide bonds. Each chain comprises a variable region (an alpha chain variable domain and a beta chain variable domain) and a constant region (an alpha chain constant region and a beta chain constant region). The variable domain is located distal to the cell membrane, and the variable domain interacts with an antigen. The constant region is located proximal to the cell membrane. The TCR can further comprise a transmembrane region and a short cytoplasmic tail. As used herein, the term “constant region” includes the transmembrane region and cytoplasmic tail as well as (when present) the traditional “constant region.”

[0091] The variable domain can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each alpha chain variable domain and beta chain variable domain comprises three CDRs and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Each variable domain comprises a binding domain that interacts with an antigen. While all three CDRs on each chain are involved in antigen binding, CDR3 is considered the primary antigen binding region, while CDR1 and CDR2 are considered to primarily recognize the HLA molecule.

[0092] The term "TCR" also includes, unless otherwise indicated, antigen-binding fragments or antigen-binding portions of any of the TCRs disclosed herein, and includes monovalent and bivalent fragments or portions, as well as single chain TCRs, unless otherwise indicated. The term "TCR" is not limited to naturally occurring TCRs that are bound to the surface of a T cell. As used herein, the term "TCR" further refers to a TCR expressed on the surface of a cell other than a T cell (e.g., a cell that naturally expresses or is modified to express CD4 as described herein) or a TCR that is not contained within a cell membrane (e.g., an isolated TCR or a soluble TCR) as described herein.

[0093] An "antigen-binding molecule," "portion of a TCR," or "TCR fragment" refers to any portion of a TCR that is less than the whole. An antigen-binding molecule can include antigenic CDRs.

[0094] An "antigen" refers to any molecule, such as a peptide, that elicits an immune response or is capable of being bound by a TCR. As used herein, an "epitope" refers to a portion of a polypeptide that elicits an immune response or is capable of being bound by a TCR. The immune response can involve the production of antibodies or the activation of specific immune-competent cells, or both. Those skilled in the art will readily understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. An antigen and / or epitope can be expressed endogenously, i.e., by genomic DNA, or can be expressed recombinantly. An antigen and / or epitope can be specific to a particular tissue, such as a diseased cell, e.g., a cancer cell, or it can be widely expressed. Furthermore, fragments of larger molecules can serve as antigens. In an aspect, the antigen is a tumor antigen. An epitope can exist within a longer polypeptide (e.g., a protein), or an epitope can exist as a fragment of a longer polypeptide. In some aspects, an epitope is complexed with a major histocompatibility complex (MHC) (also referred to herein as complexed with an HLA molecule (e.g., an HLA class 1 molecule)).

[0095] The term "autologous" refers to any material derived from the same individual, after which the material is reintroduced into the individual. For example, autologous T cell therapy includes administration of T cells to a subject that are isolated from the same subject. The term "allogeneic" refers to any material derived from one individual, after which the material is introduced into another individual of the same species. For example, allogeneic T cell transplantation includes administration of T cells to a subject that are obtained from a donor other than the subject.

[0096] "Cancer" refers to a broad group of various disorders characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include a tumor. Examples of cancers that can be treated by the methods of the present application include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, and other white blood cell malignancies. In some aspects, the methods of the present application can be used to reduce tumor size of a tumor derived from, for example, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, uterine cancer, lung cancer, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), esophageal cancer, small bowel cancer, urethral cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), bladder cancer, kidney or ureter cancer, renal pelvis cancer, glioma, squamous cell carcinoma, and combinations of said cancers. A particular cancer can be responsive to chemotherapy or radiation therapy, or the cancer can be refractory. Refractory cancer refers to a cancer that cannot be treated with surgical intervention, and the cancer is initially unresponsive or becomes unresponsive over time to chemotherapy or radiation therapy.

[0097] As used herein, the term "progression-free survival" can be abbreviated as PFS, and refers to the time from the treatment date to the date of disease progression according to the revised IWG response criteria for malignant lymphoma or death from any cause.

[0098] The term "overall survival" can be abbreviated as OS, and is defined as the time from the treatment date to the date of death.

[0099] As used herein, the term "infection" refers to any type of invasion of a foreign substance into one or more tissues of the body. The term "infection" includes, but is not limited to, infections caused by viruses (including viroids and prions), bacteria, fungi, parasites, and any combination thereof.

[0100] As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells or B cells. NK cells are a type of cytotoxic (cytotoxic / cell toxic) lymphocytes that represent a major component of the innate immune system. NK cells repel tumors and virus-infected cells. It works through a process called apoptosis or programmed cell death. They are called "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without the involvement of antibodies). T cell receptors (TCRs) distinguish T cells from other lymphocyte types. The thymus is a special organ of the immune system that is primarily responsible for the maturation of T cells. There are six types of T cells, namely: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) stem cell memory T SCM Central memory T cells, like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express high amounts of CD95, IL-2Rβ, CXCR3, and LFA-1 and display many functional attributes unique to memory cells); (ii) central memory T cells CM cells express L-selectin and CCR7, they secrete IL-2 but not IFNγ or IL-4, and (iii) effector memory T EM B cells, however, do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Tregs, suppressor T cells or CD4+CD25+ regulatory T cells), natural killer T cells (NKTs), and γδT cells. B cells, on the other hand, play a major role in humoral immunity (with the involvement of antibodies). B cells produce antibodies and antigens, function as antigen-presenting cells (APCs), and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells form in the bone marrow, hence their name.

[0101] The terms“modified” and“mutated” when used herein in reference to a nucleotide or amino acid sequence refer to a change in the sequence relative to a wild-type sequence or a particular reference sequence. Unless otherwise specified, the terms“modified” and“mutated” do not require steps in the process used to make the modified or mutated sequence (e.g., a modified beta chain sequence). Rather, these terms indicate that the modified or mutated sequence has a variation relative to a reference sequence (e.g., a wild-type sequence). For example, a DR beta chain comprising a substitution mutation at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 does not require that a wild-type DR beta chain has been physically altered to arrive at the DR beta chain; rather, when properly aligned, the DR beta chain comprises an amino acid residue at the position (residue 114) that is different from the amino acid residue at the corresponding position in a wild-type or reference DR beta chain.

[0102] As used herein, the term“any amino acid” refers to any known amino acid. An amino acid is an organic compound that contains (i) an amine (-NH2) functional group, (ii) a carboxyl (-COOH)_ functional group, and (iii) a side chain (R group), where the side chain is specific to each amino acid. This includes, but is not limited to, any naturally occurring amino acid as well as any modification and variant thereof. There are approximately 500 naturally occurring amino acids, of which 20 are encoded by the genetic code. Amino acids with positively charged side chains include arginine (Arg; R), histidine (His; H), and lysine (Lys; K). Amino acids with negatively charged side chains include aspartic acid (Asp; D) and glutamic acid (Glu; E). Amino acids with polar uncharged side chains include serine (Ser; S), threonine (Thr; T), glutamine (Gln; Q), and asparagine (Asn; N). Amino acids with hydrophobic side chains include alanine (Ala; A), isoleucine (lie; I), leucine (Leu; L), methionine (Met; M), phenylalanine (Phe; F), valine (Val; V), tryptophan (Trp; W), tyrosine (Tyr; Y). Tryptophan (Trp; W), tyrosine (Tyr; Y), and methionine (Met; M) can also be classified as polar and / or amphipathic amino acids, as these amino acids are often found on the surface of a protein or lipid membrane. Additional amino acids include cysteine (Cys; C), selenocysteine (Sec; U), glycine (Gly; G), and proline (Pro; P).

[0103] As used herein, “at a position corresponding to” is used as a means to identify a particular amino acid residue (e.g., a particular amino acid position) in a polynucleotide or a particular nucleic acid (e.g., a particular nucleic acid position) in a polypeptide. The position can be determined by aligning the sequence in question with a reference sequence properly. One of skill in the art will readily understand how to align sequences to determine relative positions. For example, various alignment tools are available online, including but not limited to “Clustal Omega Multiple Sequence Alignment” available at www.ebi.ac.uk (last accessed May 25, 2019).

[0104] The term “genetically engineered” or “engineered” refers to a method of modifying the genome of a cell, including but not limited to deletion of a coding or non-coding region or portion thereof, or insertion of a coding region or portion thereof. In some aspects, the modified cell is a lymphocyte, such as a T cell expressing CD4 or a modified cell, which can be obtained from a patient or a donor. The cell can be modified to express an exogenous construct, such as a T cell receptor (TCR) as disclosed herein, which is incorporated into the cell genome. In some aspects, the cell is modified to express CD4.

[0105] “Immune response” refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that result in the selective targeting, binding, impairment, destruction, and / or elimination of invading pathogens, cells or tissues infected with a pathogen, cancer cells or other abnormal cells, or (in the case of autoimmune or pathological inflammation) normal body cells or tissues in a vertebrate.

[0106] The term “immunotherapy” refers to the treatment of a subject having a disease or at risk of contracting a disease or suffering a recurrence of a disease by a method that includes inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy can include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation.

[0107] The cells used in the immunotherapy described herein can be from any source known in the art. For example, T cells can be differentiated in vitro from a population of hematopoietic stem cells, or the T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, the T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from unit blood collected from a subject using any number of techniques known to those of skill in the art, such as FICOLL TM separation and / or apheresis). Additional methods of isolating T cells for use in T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated by reference herein in its entirety. The immunotherapy can also include administering to the subject a modified cell, wherein the modified cell expresses a CD4 and a TCR disclosed herein. In some aspects, the modified cell is not a T cell.

[0108] As used herein, a“patient” includes any human having a cancer (e.g., a lymphoma or a leukemia). The terms“subject” and“patient” are used interchangeably herein.

[0109] The terms“peptide,”“polypeptide,” and“protein” are used interchangeably and refer to a compound composed of amino acid residues connected to one another by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids of a sequence that can make up a protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids connected to one another by peptide bonds. As used herein, the terms refer to both short chains, e.g., often referred to in the art as peptides, oligopeptides and oligomers, as well as longer chains often referred to in the art as proteins, of which there are many types. A“polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes a natural, recombinant, synthetic, or a combination thereof.

[0110] As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, wherein the binding mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, such as the T cell receptor (TCR) / CD4 complex, that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell. A "stimulatory ligand" is a molecule that, when present on an antigen presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) can specifically bind to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include, but are not limited to, MHC class II molecules loaded with peptide, anti-CD4 antibodies, anti-CD28 antibodies, anti-CD2 antibodies, and anti-CD3 antibodies.

[0111] "treatment" or "treating" a subject refers to any type of intervention or process performed on the subject, or administration of an active agent to the subject, with the objective of reversing, alleviating, improving, inhibiting, slowing down or preventing the onset of the symptoms, complications or condition, or the progression, development, severity or recurrence of a disease-related biochemical indicia. In one aspect, "treatment" or "treating" includes partial alleviation. In another aspect, "treatment" or "treating" includes complete alleviation.

[0112] Use of alternatives (e.g., "or") should be understood as meaning one, the other, or both or any combination thereof. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any described or recited component.

[0113] The terms "about" or "substantially" refer to values or compositions that are within an acceptable error range for the specific value or composition determined by one of ordinary skill in the art, which is between about 1 and 10% of the value or composition. For example, "about" or "substantially" can refer to within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" or "substantially" can mean up to 10% (i.e., ±10%) of the stated value. For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (10%). Furthermore, with respect to biological systems or processes, these terms can mean up to an order of magnitude or up to five-fold of a value. When a particular value or composition is provided in the application and claims, unless otherwise stated the intent is to recite also "about" or "substantially" that particular value or composition within an acceptable error range.

[0114] Any concentration range, percentage range, ratio range, or integer range is to be understood to include also any integer or fraction within that range.

[0115] Various aspects of the application are described in more detail in the following subsections.

[0116] II. Compositions of the Disclosure

[0117] The present disclosure relates to HLA class II molecules with enhanced CD4 binding. Certain aspects of the disclosure relate to HLA class II molecules comprising a beta chain, wherein the beta chain comprises one or more mutations. In certain aspects, the one or more mutations in the beta chain increase the affinity of the beta chain for CD4. In certain aspects, the beta chain is an HLA-DR (“DR”) beta chain.

[0118] II. A. MHC class II molecules

[0119] The human leukocyte antigen (HLA) system (the major histocompatibility complex [MHC] in humans) is an important component of the immune system and is controlled by genes located on chromosome 6. It encodes cell surface molecules that specialize in presenting antigenic peptides to T cell receptors (TCRs) on T cells. (See also Overview of the Immune System). MHC molecules that present antigens (Ag) are divided into two broad classes: class I MHC molecules and class II MHC molecules.

[0120] Class II MHC molecules exist as transmembrane glycoproteins on the surface of professional antigen-presenting cells (APCs). The complete class II molecule is composed of an alpha chain and a beta chain. Three loci in the HLA complex encode MHC class II proteins: HLA-DP, HLA-DQ, and HLA-DR. T cells that express the CD4 molecule react with class II MHC molecules. These lymphocytes generally have effector and helper functions and activate responses to eliminate self cells infected with intracellular pathogens or to destroy extracellular parasites and help other T cells, such as CD8 T cells. Because only professional APCs express class II MHC molecules, only these cells present antigens to CD4 T cells (CD4 binds to nonpolymorphic portions of the alpha-2 and beta-2 domains of the alpha and beta chains of MHC class II molecules, respectively).

[0121] In some aspects, the HLA class II alpha and beta chains are selected from HLA-DP, HLA-DQ, and HLA-DR alleles. In certain aspects, the HLA class II beta chain is an HLA-DR allele. In certain aspects, the HLA class II alpha chain is an HLA-DR allele.

[0122] Many HLA-DR alleles are known in the art, and any known allele can be used in the present disclosure. Examples of HLA-DR a chain and β chain alleles are shown in Table 1. An updated list of HLA alleles is available at hla.alleles.org / (last accessed July 10, 2019).

[0123] II.A.1. MHC Class II β Chain

[0124] In certain aspects, the HLA Class II molecule comprises a DR β chain, wherein the DR β chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1. Any amino acid other than leucine can be present at a position corresponding to amino acid residue 114 of SEQ ID NO: 1. In some aspects, the amino acid other than leucine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 is an amino acid selected from the group consisting of alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 is alanine. In certain aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 is valine. In certain aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 is isoleucine. In certain aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 is methionine. In certain aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 is phenylalanine. In certain aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 is tyrosine. In certain aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 is tryptophan.

[0125] In some embodiments, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 is composed of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects, at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 is composed of a series, e.g., at least 2, at least 3, at least 4, or at least 5 amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

[0126] In certain aspects, the HLA class II molecule comprises a DR β chain, wherein the DR β chain comprises an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1. Any amino acid other than valine can be present at a position corresponding to amino acid residue 143 of SEQ ID NO: 1. In some aspects, the amino acid other than valine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1 is an amino acid selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1 is alanine. In certain aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1 is isoleucine. In certain aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1 is leucine. In certain aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1 is methionine. In certain aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1 is phenylalanine. In certain aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1 is tyrosine. In certain aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1 is tryptophan.

[0127] In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1 is composed of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects, at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1 is composed of a series, e.g., at least 2, at least 3, at least 4, or at least 5 amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

[0128] In certain aspects, the HLA class II molecule comprises a DRP chain, wherein the DRP chain comprises an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1. Any amino acid other than serine can be present at a position corresponding to amino acid residue 118 of SEQ ID NO: 1. In some aspects, the amino acid other than serine is an amino acid comprising a charged side chain. In certain aspects, the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1 is an amino acid selected from the group consisting of arginine, histidine, and lysine. In certain aspects, the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1 is arginine. In certain aspects, the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1 is histidine. In certain aspects, the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1 is lysine.

[0129] In some aspects, the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1 is composed of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects, at least one of the more than one amino acids comprises a charged side chain. In certain aspects, the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1 is composed of a series, e.g., at least 2, at least 3, at least 4, or at least 5 amino acids, wherein each of the series of amino acids comprises a charged side chain.

[0130] In certain aspects, the HLA class II molecule comprises a DR β chain, wherein the DR β chain comprises an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1. Any amino acid other than threonine can be present at the position corresponding to amino acid residue 157 of SEQ ID NO: 1. In some aspects, the amino acid other than threonine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 is an amino acid selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 is alanine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 is valine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 is isoleucine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 is leucine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 is methionine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 is phenylalanine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 is tyrosine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 is tryptophan.

[0131] In some aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects, at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 consists of a series of, e.g., at least 2, at least 3, at least 4, or at least 5 amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

[0132] In certain aspects, the HLA class II molecule comprises a DRP chain, wherein the DRP chain comprises an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1. Any amino acid other than lysine can be present at a position corresponding to amino acid residue 139 of SEQ ID NO: 1. In some aspects, the amino acid other than lysine is an amino acid comprising a polar uncharged side chain. In certain aspects, the amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1 is an amino acid selected from serine, threonine, and glutamine. In certain aspects, the amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1 is serine. In certain aspects, the amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1 is threonine. In certain aspects, the amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1 is glutamine.

[0133] In some aspects, the amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1 is composed of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects, at least one of the more than one amino acids comprises a polar uncharged side chain. In certain aspects, the amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1 is composed of a series, e.g., at least 2, at least 3, at least 4, or at least 5 amino acids, wherein each of the series of amino acids comprises a polar uncharged side chain.

[0134] In certain aspects, the HLA class II molecule comprises a DRP chain, wherein the DRP chain comprises an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1. Any amino acid other than glycine can be present at the position corresponding to amino acid residue 146 of SEQ ID NO: 1. In some aspects, the amino acid other than glycine is an amino acid comprising a polar uncharged side chain. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 1 is an amino acid selected from serine, asparagine, threonine, and glutamine. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 1 is serine. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 1 is asparagine. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 1 is threonine. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 1 is glutamine.

[0135] In some aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 1 is composed of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects, at least one of the more than one amino acids comprises a polar uncharged side chain. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 1 is composed of a series, e.g., at least 2, at least 3, at least 4, or at least 5 amino acids, wherein each of the series of amino acids comprises a polar uncharged side chain.

[0136] In certain aspects, the HLA class II molecule comprises a DR β chain, wherein the DR β chain comprises an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1. Any amino acid other than threonine can be present at the position corresponding to amino acid residue 163 of SEQ ID NO: 1. In some aspects, the amino acid other than threonine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 is an amino acid selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 is alanine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 is valine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 is isoleucine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 is leucine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 is methionine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 is phenylalanine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 is tyrosine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 is tryptophan.

[0137] In some aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects, at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 consists of a series of, e.g., at least 2, at least 3, at least 4, or at least 5 amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

[0138] In certain aspects, the HLA class II molecule comprises a DRP chain, wherein the DRP chain comprises an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1. Any amino acid other than valine can be present at the position corresponding to amino acid residue 164 of SEQ ID NO: 1. In some aspects, the amino acid other than valine is an amino acid comprising a polar uncharged side chain. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 1 is an amino acid selected from serine, asparagine, threonine, and glutamine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 1 is serine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 1 is asparagine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 1 is threonine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 1 is glutamine.

[0139] In some aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 1 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects, at least one of the more than one amino acids comprises a polar uncharged side chain. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 1 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5 amino acids, wherein each of the series of amino acids comprises a polar uncharged side chain.

[0140] In certain aspects of the disclosure, the MHC class II molecule comprises a DRP chain that contains more than one substitution mutation relative to a wild-type DRP chain. In certain aspects, the DRP chain comprises at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, at least eight mutations, at least nine mutations, or at least ten mutations relative to a wild-type DRP chain.

[0141] In certain aspects, the DRP chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 and an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1.

[0142] In certain aspects, the DR beta chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, and (d) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1.

[0143] In certain aspects, the DR beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1; an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1; and at least two of the following: (i) an amino acid other than leucine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1; (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1; (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1; (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1; and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0144] In certain aspects, the DR beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1; an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1; and at least three of the following: (i) an amino acid other than leucine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1; (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1; (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1; (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1; and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0145] In certain aspects, the DR β chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1; an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1; and at least four of the following: (i) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1; (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1; (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1; (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1; and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0146] In certain aspects, the DR β chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, and (d) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1; and at least one of the following: (i) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1, (ii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1, (iii) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1, and (iv) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0147] In certain aspects, the DR beta chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, and (d) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1; and at least two of the following: (i) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1, (ii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1, (iii) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1, and (iv) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0148] In certain aspects, the DR beta chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, and (d) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1; and at least three of the following: (i) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1, (ii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1, (iii) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1, and (iv) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0149] In certain aspects, the DR β chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, (d) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1, (e) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1, (f) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1, (g) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1, and (h) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1.

[0150] In certain aspects, the DR β chain comprises (a) tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (b) methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (c) histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, and (d) isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1.

[0151] In some aspects, each of (i) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (ii) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, or an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 and an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1 is an amino acid comprising a hydrophobic side chain.

[0152] In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 1 is selected from the group consisting of alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan; (ii) the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 1 is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; (iii) the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 1 is selected from the group consisting of arginine, histidine, and lysine; and / or (iv) the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 is selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0153] In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 1 is selected from the group consisting of alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan; (ii) the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 1 is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; (iii) the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 1 is selected from the group consisting of arginine, histidine, and lysine; (iv) the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 1 is selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; (v) the amino acid other than lysine at the position corresponding to amino acid residue 139 of SEQ ID NO: 1 is selected from the group consisting of serine, threonine, and glutamine; (vi) the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 1 is selected from the group consisting of serine, asparagine, threonine, and glutamine; (vii) the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 1 is selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; and / or (viii) the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 1 is selected from the group consisting of serine, asparagine, threonine, and glutamine.

[0154] In certain aspects, the DR beta chain described herein has increased affinity for a CD4 protein compared to a reference HLA class II molecule. In some aspects, the reference HLA class II molecule is an HLA class II molecule having a wild-type DR beta chain. In some aspects, the reference HLA class II molecule is an HLA class II molecule having a DR beta chain comprising (i) a leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 and / or (ii) a valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1. In some aspects, the reference HLA class II molecule is an HLA class II molecule having a DR beta chain comprising (i) a leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (ii) a valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (iii) a serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1, and (iv) a threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1.

[0155] In some aspects, the increased affinity for CD4 is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 1000-fold, at least about 1500-fold, at least about 2000-fold, at least about 2500-fold, at least about 3000-fold, at least about 3500-fold, at least about 4000-fold, at least about 4500-fold, or at least about 4000-fold of the affinity for CD4 of a reference HLA class II molecule.

[0156] In some aspects, the increased affinity for CD4 is at least about 1.5-fold to at least about 5000-fold, 1.5-fold to at least about 4000-fold, 1.5-fold to at least about 3000-fold, 1.5-fold to at least about 2000-fold, 1.5-fold to at least about 1000-fold, 10-fold to at least about 5000-fold, 10-fold to at least about 4000-fold, 10-fold to at least about 3000-fold, 10-fold to at least about 2000-fold, 10-fold to at least about 1000-fold, 10-fold to at least about 900-fold, 10-fold to at least about 800-fold, 10-fold to at least about 700-fold, 10-fold to at least about 600-fold, 10-fold to at least about 500-fold, 10-fold to at least about 400-fold, 10-fold to at least about 300-fold, 10-fold to at least about 200-fold, 10-fold to at least about 100-fold, 100-fold to at least about 5000-fold, 100-fold to at least about 4000-fold, 100-fold to at least about 3000-fold, 100-fold to at least about 2000-fold, 100-fold to at least about 1000-fold, 100-fold to at least about 900-fold, 100-fold to at least about 800-fold, 100-fold to at least about 700-fold, 100-fold to at least about 600-fold, 100-fold to at least about 500-fold, 100-fold to at least about 400-fold, 100-fold to at least about 300-fold, or 100-fold to at least about 200-fold, of the reference HLA class II molecule’s affinity for CD4.

[0157] In certain aspects, the DR β chain comprises an allele selected from the group consisting of: HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*06, HLA-DRB1*07, HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*10, HLA-DRB1*11, HLA-DRB1*12, HLA-DRB1*13, HLA-DRB1*14, HLA-DRB1*15, or HLA-DRB1*16 allele. In some aspects, the DR β chain comprises a HLA-DRB1*01 allele. In particular aspects, the DR β chain comprises a HLA-DRB1*01:01 allele.

[0158] In some aspects, DRβ chain contains the following genes: DRB1*01:01:01、DRB1*01:01:02、DRB1*01:01:03、DRB1*01:01:04、DRB1*01:01:05、DRB1*01:01:06、DRB1*01:01:07、DRB1 *01:01:08、DRB1*01:01:09、DRB1*01:01:10、DRB1*01:01:11、DRB1*01:01:12、DRB1*01:01:13、DRB1*01:01:14、DRB1*01:01:15、DRB1*01:01:16、DRB1*01:01:16 B1*01:01:17、DRB1*01:01:18、DRB1*01:01:19、DRB1*01:01:20、DRB1*01:01:21、DRB1*01:01:22、DRB1*01:01:23、DRB1*01:01:24、DRB1*01:01:25、 DRB1*01:01:26、DRB1*01:01:27、DRB1*01:01:28、DRB1*01:01:29、DRB1*01:01:30、DRB1*01:01:31、DRB1*01:01:32、DRB1*01:01:33、DRB1*01:02:0 1:01、DRB1*01:02:01:02、DRB1*01:02:02、DRB1*01:02:03、DRB1*01:02:04、DRB1*01:02:05、DRB1*01:02:06、DRB1*01:02:07、DRB1*01:02:08、DRB1*01:02:09、DRB1*01:02:10、DRB1*01:02:11、DRB1*01:02:12、DRB1*01:02:13、DRB1*01:03:01、DRB1*01:03:02、DRB1*01:03:03、DRB1*01:03:04、DRB1*01:036、DRB1*01:03:07、DRB1*01:03:08、DRB1*01:03:09、DRB1*01:02:10、DRB1*01:02:11、DRB1*01:02:12、DRB1*01:02:13、DRB1*01:0 B1*01:04、DRB1*01:05、DRB1*01:06、DRB1*01:07、DRB1*01:08、DRB1*01:09、DRB1*01:10、DRB1*01:100、DRB1*01:11:01、DRB1*01:11:02、DRB1*01:10 2、DRB1*01:13、DRB1*01:14、DRB1*01:15、DRB1*01:16、DRB1*01:17、DRB1*01:18:01、DRB1*01:18:02、DRB1*01:19、DRB1*01:20:01、DRB1*01:20:02、DRB1*01:21, DRB1*01:22, DRB1*01:23, DRB1*01:24:01, DRB1*01:24:02, DRB1*01:25, DRB1*01:26, DRB1*01:27, DRB1*01:28, DRB1*01:29:01, DRB1*01:29:02, DRB1*01:30, DRB1*01:31, DRB1*01:32, DRB1*01:33N, DRB1*01:34, DRB1*01:35, DRB1*01:36, DRB1*01:37, DRB1*01:38, DRB1*01:39N, DRB1*01:40N, DRB1*01:41, DRB1*01:42, DRB1*01:43, DRB1*01:44:01, DRB1*01:44:02, DRB1*01:45, DRB1*01:46, DRB1*01:47, DRB1*01:48, DRB1*01:49, DRB1*01:50, DRB1*01:51, DRB1*01:52N, DRB1*01:53, DRB1*01:54, DRB1*01:55, DRB1*01:56, DRB1*01:57, DRB1*01:58, DRB1*01:59, DRB1*01:60, DRB1*01:61, DRB1*01:62N, DRB1*01:63, DRB1*01:64, DRB1*01:65:01, DRB1*01:65:02, DRB1*01:66, DRB1*01:67, DRB1*01:68N, DRB1*01:69, DRB1*01:70, DRB1*01:71, DRB1*01:72, DRB1*01:73, DRB1*01:74, DRB1*01:75, DRB1*01:76, DRB1*01:77, DRB1*01:78, DRB1*01:79, DRB1*01:80, DRB1*01:81, DRB1*01:82, DRB1*01:83, DRB1*01:84, DRB1*01:85, DRB1*01:86, DRB1*01:87, DRB1*01:88, DRB1*01:89, DRB1*01:90, DRB1*01:91Q, DRB1*01:92, DRB1*01:93, DRB1*01:94, DRB1*01:95, DRB1*01:96, DRB1*01:97, DRB1*01:98, DRB1*01:99, DRB1*03:01:01:01, DRB1*03:01:01:02, DRB1*03:01:01:03,DRB1*03:01:02、DRB1*03:01:03、DRB1*03:01:04、DRB1*03:01:05、DRB1*03:01:06、DRB1*03:01:07、DRB1*03:01:08、DRB1*03:01:09、DRB1*03:01:10、DRB1*03:01:11、DRB1*03:01:12、DRB1*03:01:13、DRB1*03:01:14、DRB1*03:01:15、DRB1*03:01:16、DRB1*03:01:17、DRB1*03:01:18、DRB1*03:01:1 9、DRB1*03:01:20、DRB1*03:01:21、DRB1*03:01:22、DRB1*03:01:23、DRB1*03:01:24、DRB1*03:01:25、DRB1*03:01:26、DRB1*03:01:27、DRB1*03:01:28、DRB1*03:02:01、DRB1*03:02:02、DRB1*03:02:03、DRB1*03:03、DRB1*03:04:01、DRB1*03:04:02、DRB1*03:05:01、DRB1*03:05:02、DRB1*03:05:03 、DRB1*03:06、DRB1*03:07:01、DRB1*03:07:02、DRB1*03:08、DRB1*03:09、DRB1*03:10、DRB1*03:100:01、DRB1*03:100:02、DRB1*03:10、DRB1*03:10 2、DRB1*03:103、DRB1*03:104、DRB1*03:105、DRB1*03:106、DRB1*03:107、DRB1*03:108、DRB1*03:109、DRB1*03:110、DRB1*03:111、DRB1*03:112、DRB1*03:112 1*03:113、DRB1*03:114、DRB1*03:115、DRB1*03:116、DRB1*03:117、DRB1*03:118、DRB1*03:119、DRB1*03:11:01、DRB1*03:12、DRB1*03:120、DRB1*03:121、DRB1*03:122、DRB1*03:123、DRB1*03:124、DRB1*03:125、DRB1*03:126、DRB1*03:127、DRB1*03:128、DRB1*03:129、DRB1*03:130、DRB1*03:131、DRB1*03:132, DRB1*03:133, DRB1*03:134, DRB1*03:135, DRB1*03:136, DRB1*03:137, DRB1*03:138, DRB1*03:139, DRB1*03:13:01, DRB1*03:13:02, DRB1*03:14, DRB1*03:140, DRB1*03:141, DRB1*03:142, DRB1*03:143, DRB1*03:144, DRB1*03:145, DRB1*03:146, DRB1*03:147, DRB1*03:148, DRB1*03:149, DRB1*03:150, DRB1*03:151, DRB1*03:152, DRB1*03:153, DRB1*03:154, DRB1*03:155, DRB1*03:156N, DRB1*03:157, DRB1*03:158, DRB1*03:15:01, DRB1*03:15:02, DRB1*03:16, DRB1*03:17, DRB1*03:18, DRB1*03:19, DRB1*03:20, DRB1*03:21, DRB1*03:22, DRB1*03:23, DRB1*03:24, DRB1*03:25:01, DRB1*03:25:02, DRB1*03:26, DRB1*03:27, DRB1*03:28, DRB1*03:29, DRB1*03:30, DRB1*03:31, DRB1*03:32, DRB1*03:33, DRB1*03:34, DRB1*03:35, DRB1*03:36, DRB1*03:37, DRB1*03:38, DRB1*03:39, DRB1*03:40, DRB1*03:41:01, DRB1*03:41:02, DRB1*03:42, DRB1*03:43, DRB1*03:44, DRB1*03:45, DRB1*03:46, DRB1*03:47, DRB1*03:48, DRB1*03:49, DRB1*03:50, DRB1*03:51, DRB1*03:52, DRB1*03:53, DRB1*03:54, DRB1*03:55, DRB1*03:56, DRB1*03:57, DRB1*03:58, DRB1*03:59, DRB1*03:60, DRB1*03:61, DRB1*03:62, DRB1*03:63, DRB1*03:64, DRB1*03:65, DRB1*03:66, DRB1*03:67N,DRB1*03:68N, DRB1*03:69, DRB1*03:70, DRB1*03:71 :01, DRB1*03:71 :02, DRB1*03:72, DRB1*03:73, DRB1*03:74, DRB1*03:75, DRB1*03:76, DRB1*03:77, DRB1*03:78, DRB1*03:79, DRB1*03:80, DRB1*03:81, DRB1*03:82, DRB1*03:83, DRB1*03:84, DRB1*03:85, DRB1*03:86, DRB1*03:87, DRB1*03:88, DRB1*03:89, DRB1*03:90, DRB1*03:91, DRB1*03:92, DRB1*03:93, DRB1*03:94, DRB1*03:95, DRB1*03:96, DRB1*03:97, DRB1*03:98, DRB1*03:99, DRB1*04:01 :01 :01, DRB1*04:01 :01 :02, DRB1*04:01 :01 :03, DRB1*04:01 :02, DRB1*04:01 :03, DRB1*04:01 :04, DRB1*04:01 :05, DRB1*04:01 :06, DRB1*04:01 :07, DRB1*04:01 :08, DRB1*04:01 :09, DRB1*04:01 :10, DRB1*04:01 :11, DRB1*04:01 :12, DRB1*04:01 :13, DRB1*04:01 :14, DRB1*04:01 :15, DRB1*04:01 :16, DRB1*04:01 :17, DRB1*04:01 :18, DRB1*04:01 :19, DRB1*04:01 :20, DRB1*04:01 :21, DRB1*04:02:01, DRB1*04:02:02, DRB1*04:02:03, DRB1*04:02:04, DRB1*04:02:05, DRB1*04:02:06, DRB1*04:03:01 :01, DRB1*04:03:01 :02, DRB1*04:03:02, DRB1*04:03:03, DRB1*04:03:04, DRB1*04:03:05, DRB1*04:03:06, DRB1*04:03:07, DRB1*04:03:08, DRB1*04:03:09, DRB1*04:03:10, DRB1*04:03:11, DRB1*04:03:12, DRB1*04:03:13, DRB1*04:03:14,DRB1*04:03:15, DRB1*04:04:01, DRB1*04:04:02, DRB1*04:04:03, DRB1*04:04:04, DRB1*04:04:05, DRB1*04:04:06, DRB1*04:04:07, DRB1*04:04:08, DRB1*04:04:09, DRB1*04:04:10, DRB1*04:04:11, DRB1*04:04:12, DRB1*04:04:13, DRB1*04:04:14, DRB1*04:04:15, DRB1*04:05:01:01, DRB1*04:05:01:02, DRB1*04:05:01:03, DRB1*04:05:02, DRB1*04:05:03, DRB1*04:05:04, DRB1*04:05:05, DRB1*04:05:06, DRB1*04:05:07, DRB1*04:05:08, DRB1*04:05:09, DRB1*04:05:10, DRB1*04:05:11, DRB1*04:05:13, DRB1*04:05:14, DRB1*04:05:15, DRB1*04:05:16, DRB1*04:05:17, DRB1*04:05:18, DRB1*04:05:19, DRB1*04:05:20, DRB1*04:06:01, DRB1*04:06:02, DRB1*04:06:03, DRB1*04:06:04, DRB1*04:06:05, DRB1*04:06:06, DRB1*04:06:07, DRB1*04:07:01:01, DRB1*04:07:01:02, DRB1*04:07:02, DRB1*04:07:03, DRB1*04:07:04, DRB1*04:07:05, DRB1*04:07:06, DRB1*04:08:01, DRB1*04:08:02, DRB1*04:08:03, DRB1*04:08:04, DRB1*04:09, DRB1*04:100, DRB1*04:101, DRB1*04:102, DRB1*04:103, DRB1*04:104, DRB1*04:105:01, DRB1*04:105:02, DRB1*04:106, DRB1*04:107, DRB1*04:108, DRB1*04:109, DRB1*04:10:01, DRB1*04:10:02, DRB1*04:10:03, DRB1*04:110, DRB1*04:111,DRB1*04:112, DRB1*04:113, DRB1*04:114, DRB1*04:115, DRB1*04:116, DRB1*04:117, DRB1*04:118, DRB1*04:119N, DRB1*04:11:01, DRB1*04:11:02, DRB1*04:11:03, DRB1*04:11:04, DRB1*04:11:05, DRB1*04:12, DRB1*04:120N, DRB1*04:121, DRB1*04:122, DRB1*04:123, DRB1*04:124, DRB1*04:125, DRB1*04:126, DRB1*04:127, DRB1*04:128, DRB1*04:129, DRB1*04:13, DRB1*04:130, DRB1*04:131:01, DRB1*04:131:02, DRB1*04:132, DRB1*04:133, DRB1*04:134, DRB1*04:135, DRB1*04:136, DRB1*04:137, DRB1*04:138, DRB1*04:139, DRB1*04:14, DRB1*04:140, DRB1*04:141, DRB1*04:142N, DRB1*04:143, DRB1*04:144, DRB1*04:145, DRB1*04:146, DRB1*04:147, DRB1*04:148, DRB1*04:149, DRB1*04:15, DRB1*04:150, DRB1*04:151, DRB1*04:152, DRB1*04:153, DRB1*04:154, DRB1*04:155, DRB1*04:156, DRB1*04:157N, DRB1*04:158N, DRB1*04:159, DRB1*04:16, DRB1*04:160, DRB1*04:161, DRB1*04:162, DRB1*04:163, DRB1*04:164, DRB1*04:165, DRB1*04:166, DRB1*04:167, DRB1*04:168, DRB1*04:169, DRB1*04:170, DRB1*04:171, DRB1*04:172, DRB1*04:173, DRB1*04:174, DRB1*04:175, DRB1*04:176, DRB1*04:177, DRB1*04:178N, DRB1*04:179, DRB1*04:17:01, DRB1*04:17:02,DRB1*04:18, DRB1*04:180, DRB1*04:181, DRB1*04:182, DRB1*04:183, DRB1*04:184, DRB1*04:185, DRB1*04:186N, DRB1*04:187, DRB1*04:188, DRB1*04:189, DRB1*04:19, DRB1*04:190, DRB1*04:191, DRB1*04:192, DRB1*04:193, DRB1*04:194, DRB1*04:195, DRB1*04:196, DRB1*04:197, DRB1*04:198, DRB1*04:199, DRB1*04:20, DRB1*04:200, DRB1*04:201, DRB1*04:202, DRB1*04:203, DRB1*04:204, DRB1*04:205, DRB1*04:206, DRB1*04:207, DRB1*04:208, DRB1*04:209, DRB1*04:21, DRB1*04:210, DRB1*04:211, DRB1*04:212N, DRB1*04:213, DRB1*04:214N, DRB1*04:215, DRB1*04:216, DRB1*04:217, DRB1*04:218, DRB1*04:219, DRB1*04:22, DRB1*04:220, DRB1*04:221, DRB1*04:222, DRB1*04:223, DRB1*04:224, DRB1*04:225, DRB1*04:226:01, DRB1*04:226:02, DRB1*04:227, DRB1*04:228, DRB1*04:229, DRB1*04:23, DRB1*04:230, DRB1*04:231, DRB1*04:232, DRB1*04:233, DRB1*04:234, DRB1*04:235, DRB1*04:236, DRB1*04:237, DRB1*04:238, DRB1*04:239, DRB1*04:24, DRB1*04:240, DRB1*04:241, DRB1*04:242, DRB1*04:243, DRB1*04:244, DRB1*04:245, DRB1*04:246, DRB1*04:247N, DRB1*04:248, DRB1*04:249, DRB1*04:25, DRB1*04:250, DRB1*04:251, DRB1*04:252, DRB1*04:253,DRB1*04:254, DRB1*04:255, DRB1*04:256, DRB1*04:257, DRB1*04:258, DRB1*04:259, DRB1*04:26, DRB1*04:260, DRB1*04:261, DRB1*04:262, DRB1*04:263, DRB1*04:264N, DRB1*04:265, 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DRB1*15:01:01:02, DRB1*15:01:01:03, DRB1*15:01:01:04, DRB1*15:01:01:05, DRB1*15:01:02, DRB1*15:01:03, DRB1*15:01:04, DRB1*15:01:05, DRB1*15:01:06, DRB1*15:01:07, DRB1*15:01:08, DRB1*15:01:09, DRB1*15:01:10, DRB1*15:01:11, DRB1*15:01:12, DRB1*15:01:13, DRB1*15:01:14, DRB1*15:01:15, DRB1*15:01:16, DRB1*15:01:17, DRB1*15:01:18, DRB1*15:01:19, DRB1*15:01:20,DRB1*15:01:21, DRB1*15:01:22, DRB1*15:01:23, DRB1*15:01:24, DRB1*15:01:25, DRB1*15:01:26, DRB1*15:01:27, DRB1*15:01:28, DRB1*15:01:29, DRB1*15:01:30, DRB1*15:01:31, DRB1*15:01:32, DRB1*15:01:33, DRB1*15:01:34, DRB1*15:01:35, DRB1*15:01:36, DRB1*15:01:37, DRB1*15:01:38, DRB1*15:01:39, DRB1*15:01:40, DRB1*15:01:41, DRB1*15:02:01:01, DRB1*15:02:01:02, DRB1*15:02:01:03, DRB1*15:02:02, DRB1*15:02:03, DRB1*15:02:04, DRB1*15:02:05, DRB1*15:02:06, DRB1*15:02:07, DRB1*15:02:08, DRB1*15:02:09, DRB1*15:02:10, DRB1*15:02:11, DRB1*15:02:12, DRB1*15:02:13, DRB1*15:02:14, DRB1*15:02:15, DRB1*15:02:16, DRB1*15:02:17, DRB1*15:02:18, DRB1*15:02:19, DRB1*15:03:01:01, DRB1*15:03:01:02, DRB1*15:03:01:03, DRB1*15:03:02, DRB1*15:03:03, DRB1*15:03:04, DRB1*15:04, DRB1*15:05, DRB1*15:06:01, DRB1*15:06:02, DRB1*15:06:03, DRB1*15:06:04, DRB1*15:07:01, DRB1*15:07:02, DRB1*15:07:03, DRB1*15:08, DRB1*15:09, DRB1*15:10, DRB1*15:100, DRB1*15:101, DRB1*15:102, DRB1*15:103, DRB1*15:104:01, DRB1*15:104:02, DRB1*15:104:03, DRB1*15:105:01, DRB1*15:105:02, DRB1*15:106, DRB1*15:107,DRB1*15:108, DRB1*15:109, DRB1*15:110, DRB1*15:111, DRB1*15:112, DRB1*15:113N, DRB1*15:114, DRB1*15:115N, DRB1*15:116, DRB1*15:117, DRB1*15:118, DRB1*15:119, DRB1*15:11:01, DRB1*15:11:02, DRB1*15:12, DRB1*15:120, DRB1*15:121, DRB1*15:122, DRB1*15:123, DRB1*15:124, DRB1*15:125, DRB1*15:126, DRB1*15:127, DRB1*15:128, DRB1*15:129N, DRB1*15:13, DRB1*15:130, DRB1*15:131, DRB1*15:132, DRB1*15:133, DRB1*15:134N, DRB1*15:135, DRB1*15:136, DRB1*15:137N, DRB1*15:138N, DRB1*15:139, DRB1*15:14, DRB1*15:140, DRB1*15:141, DRB1*15:142, DRB1*15:143, DRB1*15:144, DRB1*15:145, DRB1*15:146, DRB1*15:147, DRB1*15:148N, DRB1*15:149, DRB1*15:150, DRB1*15:151, DRB1*15:152, DRB1*15:153, DRB1*15:154N, DRB1*15:155, DRB1*15:156, DRB1*15:157, DRB1*15:158, DRB1*15:159N, DRB1*15:15:01, DRB1*15:15:02, DRB1*15:15:03, DRB1*15:16, DRB1*15:160, DRB1*15:161, DRB1*15:162, DRB1*15:163N, DRB1*15:164Q, DRB1*15:165, DRB1*15:166, DRB1*15:167, DRB1*15:168, DRB1*15:169, DRB1*15:170, DRB1*15:17N, DRB1*15:18, DRB1*15:19, DRB1*15:20, DRB1*15:21, DRB1*15:22, DRB1*15:23, DRB1*15:24, DRB1*15:25, DRB1*15:26,DRB1*15:27, DRB1*15:28, DRB1*15:29, DRB1*15:30, DRB1*15:31:01, DRB1*15:31:02, DRB1*15:32, DRB1*15:33, DRB1*15:34, DRB1*15:35, DRB1*15:36, DRB1*15:37:01, DRB1*15:37:02, DRB1*15:38, DRB1*15:39, DRB1*15:40, DRB1*15:41, DRB1*15:42, DRB1*15:43, DRB1*15:44, DRB1*15:45, DRB1*15:46, DRB1*15:47, DRB1*15:48, DRB1*15:49, DRB1*15:50N, DRB1*15:51, DRB1*15:52, DRB1*15:53, DRB1*15:54, DRB1*15:55, DRB1*15:56, DRB1*15:57, DRB1*15:58, DRB1*15:59, DRB1*15:60, DRB1*15:61, DRB1*15:62, DRB1*15:63, DRB1*15:64, DRB1*15:65, DRB1*15:66:01, DRB1*15:66:02, DRB1*15:67, DRB1*15:68, DRB1*15:69, DRB1*15:70, DRB1*15:71, DRB1*15:72, DRB1*15:73, DRB1*15:74, DRB1*15:75, DRB1*15:76, DRB1*15:77, DRB1*15:78, DRB1*15:79, DRB1*15:80N, DRB1*15:81, DRB1*15:82, DRB1*15:83, DRB1*15:84, DRB1*15:85, DRB1*15:86, DRB1*15:87, DRB1*15:88, DRB1*15:89, DRB1*15:90, DRB1*15:91, DRB1*15:92, DRB1*15:93, DRB1*15:94, DRB1*15:95, DRB1*15:96, DRB1*15:97, DRB1*15:98, DRB1*15:99, DRB1*16:01:01, DRB1*16:01:02, DRB1*16:01:03, DRB1*16:01:04, DRB1*16:01:05, DRB1*16:01:06, DRB1*16:01:07, DRB1*16:01:08, DRB1*16:01:09, DRB1*16:01:10,DRB1* 16:01 : 11, DRB1* 16:01 : 12, DRB1* 16:01 : 13, DRB1* 16:01 : 14, DRB1* 16:01 : 15, DRB1* 16:01 : 16, DRB1* 16:02:01 : 01, DRB1* 16:02:01 : 02, DRB1* 16:02:01 : 03, DRB1* 16:02:02, DRB1* 16:02:03, DRB1* 16:02:04, DRB1* 16:02:05, DRB1* 16:02:06, DRB1* 16:02:07, DRB1* 16:02:08, DRB1* 16:03, DRB1* 16:04:01, DRB1* 16:04:02, DRB1* 16:05:01, DRB1* 16:05:02, DRB1* 16:07, DRB1* 16:08, DRB1* 16:09:01, DRB1* 16:09:02, DRB1* 16:10:01, DRB1* 16:10:02, DRB1* 16:11, DRB1* 16:12, DRB1* 16:13N, DRB1* 16:14, DRB1* 16:15, DRB1* 16:16, DRB1* 16:17, DRB1* 16:18, DRB1* 16:19, DRB1* 16:20, DRB1* 16:21N, DRB1* 16:22, DRB1* 16:23, DRB1* 16:24, DRB1* 16:25, DRB1* 16:26, DRB1* 16:27, DRB1* 16:28, DRB1* 16:29, DRB1* 16:30, DRB1* 16:31, DRB1* 16:32, DRB1* 16:33, DRB1* 16:34, DRB1* 16:35, DRB1* 16:36, DRB1* 16:37, DRB1* 16:38:01, DRB1* 16:38:02, DRB1* 16:39, DRB1* 16:40, DRB1* 16:41N, DRB1* 16:42, DRB1* 16:43, DRB1* 16:44, DRB1* 16:45, DRB1* 16:46, DRB1* 16:47, DRB1* 16:48, DRB1* 16:49, DRB1* 16:50, DRB1* 16:51, DRB1* 16:52, DRB1* 16:53, DRB1* 16:54, DRB1* 16:55N, DRB1* 16:56, and any combination thereof.

[0159] In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 3, wherein the DR beta chain comprises (i) a tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (ii) a methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (iii) a histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1; and (vi) an isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1. In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 3, wherein the DR beta chain comprises (i) a tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, (ii) a methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 1, (iii) a histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 1; (iv) an isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 1; (v) a threonine at a position corresponding to amino acid residue 139 of SEQ ID NO: 1; (vi) a glutamine at a position corresponding to amino acid residue 146 of SEQ ID NO: 1; (vii) a methionine at a position corresponding to amino acid residue 163 of SEQ ID NO: 1; and (vii) a threonine at a position corresponding to amino acid residue 164 of SEQ ID NO: 1. In certain aspects, the MHC class II molecule comprises a DR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 3.

[0160] II.A.2. MHC class II alpha chain

[0161] In some aspects of the disclosure, the MHC class II molecule further comprises an alpha chain. In some aspects, the alpha chain is a wild-type alpha chain. In some aspects, the alpha chain is a DR alpha chain. Any DR alpha chain can be used in the compositions and methods of the disclosure. In some aspects, the DR alpha chain comprises an HLA-DRA1*01 allele.

[0162] In certain aspects, the DRa chain is selected from the group consisting of DRA*01:01:01:01, DRA*01:01:01:02, DRA*01:01:01:03, DRA*01:01:02, DRA*01:02:01, DRA*01:02:02, DRA*01:02:03, and any combination thereof.

[0163] II.A.3. Signal Peptide

[0164] In some aspects, the DRP chain and / or the DRa chain further comprises a signal peptide. Any signal peptide known in the art can be used in the compositions and methods disclosed herein. In some aspects, the DRP chain signal peptide is the same as the DRa signal peptide. In some aspects, the DRP chain signal peptide is different from the DRa signal peptide.

[0165] In some aspects, the signal peptide is derived from a naturally occurring signal peptide. In some aspects, the signal peptide is derived from a naturally occurring DRP chain signal peptide. In some aspects, the signal peptide comprises a naturally occurring DRP chain signal peptide. In some aspects, the signal peptide is derived from a naturally occurring DRa chain signal peptide. In some aspects, the signal peptide comprises a naturally occurring DRa chain signal peptide. In some aspects, the signal peptide is derived from a fibulin light chain (FibL) signal peptide. In some aspects, the signal peptide comprises SEQ ID NO: 9. In some aspects, the signal peptide is synthetic.

[0166] II.A.4. Transmembrane Domain

[0167] In some aspects, the DRP chain and / or the DRa chain further comprises a transmembrane domain. The transmembrane domain can be of any length and of any origin. In some aspects, the transmembrane domain is at least about 1 to at least about 50 amino acids in length. In some aspects, the transmembrane domain is derived from a naturally occurring transmembrane domain. In some aspects, the transmembrane domain comprises a naturally occurring transmembrane domain. In some aspects, the transmembrane domain is derived from a naturally occurring HLA transmembrane domain. In some aspects, the transmembrane domain comprises a naturally occurring HLA transmembrane domain. In some aspects, the transmembrane domain is derived from a naturally occurring DRP chain transmembrane domain. In some aspects, the transmembrane domain comprises a naturally occurring DRP chain transmembrane domain. In some aspects, the transmembrane domain is derived from a naturally occurring DRa chain transmembrane domain. In some aspects, the transmembrane domain comprises a naturally occurring DRa chain transmembrane domain.

[0168] II.A.5. Leucine Zipper

[0169] In some aspects, the DR beta chain and / or the DR alpha chain further comprises one or more leucine zipper (LZip) sequences. Any LZip sequence known in the art can be used in the compositions and methods disclosed herein. In some aspects, the DR beta chain and / or the DR alpha chain comprises an acidic LZip (aLZip), a basic LZip (bLZip), or both. In some aspects, the one or more LZip sequences are derived from a naturally occurring LZip sequence. In some aspects, the one or more LZip sequences comprise a naturally occurring LZip sequence. In some aspects, the one or more LZip sequences are synthetic. In certain aspects, the one or more LZip sequences comprise the LZip sequence set forth in SEQ ID NO: 4 (Table 1).

[0170] II.A.6. Linker

[0171] In some aspects, the DR beta chain and / or the DR alpha chain useful in the present disclosure further comprises a linker. Any linker known in the art can be used in the compositions and methods disclosed herein. In certain aspects, the linker comprises a Gly / Ser linker. In some aspects, the linker comprises an amino acid sequence selected from the group consisting of GlySer, Gly2Ser, Gly3Ser, and Gly4Ser. In some aspects, the linker is located at the N-terminus of the extracellular domain of the DR alpha chain or the DR beta chain. In some aspects, the linker is located at the C-terminus of the extracellular domain of the DR alpha chain or the DR beta chain. In some aspects, the linker is located between the extracellular domain and the transmembrane domain of the DR alpha chain or the DR beta chain. In some aspects, the linker is located between the extracellular domain and the one or more LZip sequences of the DR alpha chain or the DR beta chain. In some aspects, the linker is located between the extracellular domain and the signal peptide of the DR alpha chain or the DR beta chain.

[0172] Any length of linker can be used in the compositions and methods disclosed herein. In some aspects, the length of the linker is at least one amino acid. In some aspects, the length of the linker is at least about 1 to at least about 100, at least about 1 to at least about 90, at least about 1 to at least about 80, at least about 1 to at least about 70, at least about 1 to at least about 60, at least about 1 to at least about 50, at least about 1 to at least about 40, at least about 1 to at least about 30, at least about 1 to at least about 20, at least about 1 to at least about 15, at least about 1 to at least about 14, at least about 1 to at least about 13, at least about 1 to at least about 12, at least about 1 to at least about 11, at least about 1 to at least about 10, at least about 1 to at least about 9, at least about 1 to at least about 8, at least about 1 to at least about 7, at least about 1 to at least about 6, at least about 1 to at least about 5, at least about 1 to at least about 4, at least about 1 to at least about 3 amino acids.

[0173] In some aspects, the length of the linker is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100 amino acids. In some aspects, the length of the linker is about 3 amino acids. In some aspects, the length of the linker is about 4 amino acids. In some aspects, the length of the linker is about 5 amino acids.

[0174] II.B. Cells

[0175] In certain aspects of the disclosure, the MHC class II molecule of the disclosure is linked to or associated with the membrane of a cell. In certain aspects, the beta chain of the MHC class II molecule is linked to or associated with the membrane of a cell. In certain aspects, the alpha chain of the MHC class II molecule is linked to or associated with the membrane of a cell. In certain aspects, the alpha chain and the beta chain of the MHC class II molecule are linked to or associated with the membrane of a cell.

[0176] Certain aspects of the disclosure relate to a cell comprising the MHC class II molecule disclosed herein. Any cell can be used in the compositions described herein. In certain aspects, the cell is a mammalian cell. In some aspects, the cell is an insect cell. In some aspects, the cell is derived from a healthy cell, e.g., a healthy fibroblast cell. In some aspects, the cell is derived from a tumor cell. Non-limiting examples of cells that can be used in the disclosure include a K562 cell, a T2 cell, a HEK293 cell, a HEK293T cell, an A375 cell, an SK-MEL-28 cell, a Me275 cell, a COS cell, a fibroblast cell, a tumor cell, or any combination thereof. In certain aspects, the cell is any of the cells disclosed in Hasan et al., Adv. Genet. Eng. 4(3): 130 (2015), incorporated by reference in its entirety.

[0177] In certain aspects, the cell is a professional APC. In certain aspects, the cell is a macrophage, a B cell, a dendritic cell, or any combination thereof.

[0178] In certain aspects, the cell lacks endogenous expression of one or more MHC class II alleles. In some aspects, the cell lacks endogenous expression of an HLA-DR allele. In some aspects, the cell lacks endogenous expression of an HLA-DR alpha chain allele. In some aspects, the cell lacks endogenous expression of an HLA-DR beta chain allele.

[0179] II.C. Soluble MHC class II molecules

[0180] In certain aspects, the MHC class II molecule is not associated with a cell membrane, e.g., the MHC class II molecule is in a soluble form. As used herein, a soluble MHC class II molecule includes any MHC class II molecule or portion thereof described herein that is not associated with a cell membrane. In certain aspects, the MHC class II molecule or portion thereof is not bound to any membrane. In some aspects, the MHC class II molecule or portion thereof is bound to an inert particle. In some aspects, the MHC class II molecule or portion thereof is bound to the membrane of an extracellular vesicle. In some aspects, the MHC class II molecule is bound to an artificial membrane or artificial surface, e.g., the surface of an array plate.

[0181] Any inert particle known in the art can be used in the compositions and methods of the present disclosure. In some aspects, the inert particle is a bead. In some aspects, the bead is a glass bead, a latex bead, a metal bead, or any combination thereof. In some aspects, the inert particle is a nanoparticle (NP). Any NP known in the art can be used in the compositions and methods of the present disclosure. In certain aspects, the nanoparticle is selected from the group consisting of polyethylene glycolated iron oxide, chitosan, dextran, gelatin, alginate, liposome, starch, branched polymer, carbon-based carrier, polylactic acid, poly(cyano)acrylate, polyethylenimine, block copolymer, polycaprolactone, SPIONS, USPIONS, Cd / Zn-selenide, or silica nanoparticles. In particular aspects, the nanoparticle is a polyethylene glycolated iron oxide nanoparticle. Non-limiting examples of nanoparticles that can be used in the compositions and methods disclosed herein include those set forth in De Jong and Borm, Int. J. Nanomedicine 3(2): 133-49 (2008) and Umeshappa et al., Nat. Commun. 10(1): 2150 (May 14, 2019), each incorporated by reference herein in their entirety.

[0182] In some aspects, the MHC class II molecule comprises a fragment of a full-length MHC class II molecule in which one or more amino acids of the transmembrane domain of the alpha chain and / or the transmembrane domain of the beta chain are deleted. In some aspects, the MHC class II molecule comprises an extracellular domain of the alpha chain (e.g., as set forth in SEQ ID NO: 6) and / or an extracellular domain of the beta chain (e.g., as set forth in SEQ ID NO: 1 or 3). In certain aspects, the MHC class II molecule comprises a DR alpha chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 6. In some aspects, the MHC class II molecule comprises a DR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0183] In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 1. In some aspects, the MHC class II molecule comprises a DR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 1. In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 3. In certain aspects, the MHC class II molecule comprises a DR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 4. In some aspects, the MHC class II molecule comprises a DR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 5. In some aspects, the MHC class II molecule comprises a DR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0184] II.D. NUCLEIC ACID MOLECULES AND VECTORS

[0185] Certain aspects of the present disclosure relate to nucleic acid molecules encoding the MHC class II molecules disclosed herein. In some aspects, the nucleic acid molecule encodes the MHC class II beta chain disclosed herein. In certain aspects, the nucleic acid molecule encoding the MHC class II beta chain comprises a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 2.

[0186] In some aspects, the nucleic acid molecule encodes a MHC class II alpha chain disclosed herein. In certain aspects, the nucleic acid molecule encoding the MHC class II alpha chain comprises a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 7.

[0187] In some aspects, the nucleic acid molecule encodes both a MHC class II alpha chain disclosed herein and a MHC class II beta chain disclosed herein. In some aspects, the sequence encoding the MHC class II alpha chain is under the control of the same promoter as the sequence encoding the MHC class II beta chain. In some aspects, the sequence encoding the MHC class II alpha chain is under the control of a first promoter and the sequence encoding the MHC class II beta chain is under the control of a second promoter.

[0188] In some aspects, the disclosure relates to a first nucleic acid molecule encoding a MHC class II beta chain disclosed herein and a second nucleic acid molecule encoding a MHC class II alpha chain disclosed herein.

[0189] Certain aspects of the disclosure relate to a vector or a set of vectors comprising a nucleic acid molecule disclosed herein. In some aspects, the vector is a viral vector. In some aspects, the vector is a viral particle or a virus. In some aspects, the vector is a mammalian vector. In some aspects, the vector is a bacterial vector.

[0190] In certain aspects, the vector is a retroviral vector. In some aspects, the vector is an adenoviral vector, a lentivirus, a Sendai virus, a baculovirus vector, an Epstein-Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, or an adeno-associated virus (AAV) vector. In particular aspects, the vector is an AAV vector. In some aspects, the vector is a lentivirus. In particular aspects, the vector is an adenoviral vector. In some aspects, the vector is a Sendai virus. In some aspects, the vector is a hybrid vector. Examples of hybrid vectors that can be used in the present disclosure can be found in Huang and Kamihira, Biotechnol. Adv. 31(2):208-23 (2103), incorporated by reference herein in its entirety.

[0191] III. Methods of the Disclosure

[0192] Certain aspects of the disclosure relate to methods of treating a disease or condition in a subject. In some aspects, the disclosure relates to methods of enhancing an immune response in a subject in need thereof.

[0193] III. A. Methods of treating a tumor

[0194] Certain aspects of the present disclosure relate to methods of treating a cancer in a subject in need thereof, comprising administering to the subject an HLA Class II molecule disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, or a cell disclosed herein.

[0195] In some aspects, the cancer is selected from melanoma, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or intraocular malignant melanoma, pancreatic cancer, skin cancer, cancer of the head or neck, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, acute or chronic leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) (including non T-cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and any combination of these cancers. In some aspects, the cancer is melanoma.

[0196] In some aspects, the cancer is recurrent. In some aspects, the cancer is refractory. In some aspects, the cancer is advanced. In some aspects, the cancer is metastatic.

[0197] In some aspects, the methods disclosed herein treat a cancer in a subject. In some aspects, the methods disclosed herein reduce the severity of one or more symptoms of a cancer. In some aspects, the methods disclosed herein reduce the size or number of tumors derived from the cancer. In some aspects, the methods disclosed herein increase the overall survival of a subject relative to a subject not provided the methods disclosed herein. In some aspects, the methods disclosed herein increase the progression-free survival of a subject relative to a subject not provided the methods disclosed herein. In some aspects, the methods disclosed herein result in a partial response in a subject. In some aspects, the methods disclosed herein result in a complete response in a subject.

[0198] Certain aspects of the present disclosure relate to methods of treating an infection in a subject in need thereof, the methods comprising administering to the subject an HLA class II molecule disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, or a cell disclosed herein. Non-limiting examples of infections that can be treated using the compositions and methods disclosed herein include infections of viruses (including viroids and prions), bacteria, fungi, parasites, or any combination thereof. In some aspects, the virus is a herpes virus, HIV, a papovavirus, a morbillivirus, a rubivirus, a human papillomavirus (HPV), human T-lymphotrophic virus 1, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, norovirus, and any combination thereof. In some aspects, the bacteria is selected from the group consisting of Streptococcus, Staphylococcus, and Escherichia coli. In some aspects, the bacterial infection is selected from the group consisting of brucellosis, Campylobacter infection, cat scratch disease, cholera, E. coli infection, gonorrhea, Klebsiella infection, Enterobacter infection, Serratia infection, Legionella infection, meningococcal infection, pertussis, plague, Pseudomonas infection, Salmonella infection, Shigellosis, typhoid fever, tularemia, anthrax, diphtheria, Enterococcus infection, Erysipelothricosis, Listeriosis, Nocardiosis, Pneumococcal infection, Staphylococcus infection, Streptococcus infection, and any combination thereof. In some embodiments, the parasitic infection is selected from the group consisting of enterobiasis, trichomoniasis, toxoplasmosis, giardiasis, cryptosporidiosis, malaria, hookworm disease, ringworm, tapeworm disease, fluke disease, and any combination thereof. In some aspects, the fungal infection is selected from the group consisting of Candida, Malassezia furfur, dermatophytes (e.g., Epidermophyton, Microsporum, and Trichophyton), or any combination thereof.

[0199] In some aspects, the methods disclosed herein comprise treating a cancer or an infection in a subject in need thereof, the methods comprising administering to the subject a cell described herein, wherein the cell comprises an MHC class II molecule disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, or any combination thereof.

[0200] In some aspects, the cell is obtained from the subject. In some aspects, the cell is obtained from a donor other than the subject.

[0201] III.B. Methods of enriching a target T cell population

[0202] Certain aspects of the present disclosure relate to methods of enriching a target T cell population obtained from a human subject. In some aspects, the methods comprise contacting the T cells with an HLA class II molecule disclosed herein. In some aspects, the methods comprise contacting the T cells with a cell (e.g., an APC) disclosed herein. In some aspects, following the contacting, the enriched T cell population comprises a greater number of T cells capable of binding to the HLA class II molecule relative to the number of T cells capable of binding to the HLA class II molecule prior to the contacting.

[0203] Some aspects of the disclosure relate to methods of selecting T cells that are capable of targeting diseased cells, e.g., tumor cells. In some aspects, the methods comprise contacting a population of isolated T cells in vitro with a complex comprising an MHC class II molecule disclosed herein and a fragment of a polypeptide, e.g., an antigen expressed by a diseased cell, e.g., a polypeptide expressed by a tumor, e.g., an epitope. In some aspects, the T cells are obtained from a human subject.

[0204] The T cells obtained from a human subject can be any T cell disclosed herein. In some aspects, the T cells obtained from a human subject are tumor infiltrating lymphocytes (TILs).

[0205] In some aspects, the methods further comprise administering the enriched T cells to a human subject. In some aspects, the subject is preconditioned prior to receiving the T cells as described herein.

[0206] All of the various aspects, aspects, and options described herein can be combined in any and all variations.

[0207] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0208] Having generally described this disclosure, a further understanding can be obtained by reference to the examples provided herein. These examples are for illustrative purposes only and are not intended to limit the disclosure.

[0209] Examples

[0210] Example 1 - Methods

[0211] Cells

[0212] Peripheral mononuclear cells were obtained by density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare LifeSciences, Marlborough, MA). The K562 cell line is a chronic myelogenous leukemia cell line with defective HLA class I / II expression. K562-based artificial APCs (aAPCs) expressing various HLA class II genes as single HLA alleles along with CD80 and CD83 have been previously reported (Butler et al., PloS One 7, e30229 (2012)). HEK293T cells were grown in DMEM supplemented with 10% FBS and 50 pg / ml gentamicin (Thermo Fisher Scientific, Waltham, MA). K562 cells were cultured in RPMI 1640 supplemented with 10% FBS and 50 pg / ml gentamicin.

[0213] Peptides

[0214] Synthetic peptides were purchased from Genscript (Piscataway, NJ) and dissolved in DMSO at 50 pg / ml.

[0215] Antibodies

[0216] The following antibodies were used for flow cytometry analysis: PE-conjugated anti-class II (9-49 (13)), APC-Cy7-conjugated anti-CD4 (RPA-T4, Biolegend, San Diego, CA), PE-conjugated anti-His tag (AD1.1.10, Abeam, Cambridge, MA), and FITC-conjugated anti-Vp22 (IMMU 546, Beckman Coulter, Brea, CA). Dead cells were distinguished with the Live / Dead Fixable Near-IR Dead Cell Stain Kit 465 (Thermo Fisher Scientific, Waltham, MA). Stained cells were analyzed with a Canto II or LSRFortessa X-20 (BD Biosciences, Franklin Lakes, NJ). Cell sorting was performed using a FACS Aria II (BD Biosciences, Franklin Lakes, NJ). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR). 44

[0217] TCR transduction into primary T cells

[0218] Using CD4 + ​T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) to purify CD4 + T cells. Purified T cells were stimulated with 200 Gy irradiated aAPC / mOKT3 at an E:T ratio of 20: 1. From the next day, activated T cells were retrovirally transduced with cloned TCR genes by centrifugation at 1,000 x g for 1 hour at 32°C for 3 consecutive days or using Retronectin coated plates (Takara Bio, Shiga, Japan). On the second day, 100 IU / ml IL-2 and 10 ng / ml IL-15 were added to the TCR transduced T cells. Medium was replenished every 2-3 days.

[0219] Staining with soluble CD4

[0220] The soluble CD4 (sCD4) gene was generated by fusing the extracellular domain of human CD4 with a 6xHis tag via a GS linker. HEK293T cells were retrovirally transduced with the sCD4 gene and culture supernatant containing sCD4 monomers was harvested. sCD4 was dimerized with a PE-labeled anti-6xHis tag mAb (AD1.1.10, Abeam, Cambridge, MA) and used. K562 cells expressing HLA class II were stained with dimerized sCD4 in the presence of goat serum for 30 minutes at room temperature. Surface HLA class II expression in K562-derived cells expressing various class II genes, respectively, was determined as shown in Figure 1E

[0221] Generation of HLA class II monomers and dimers

[0222] The extracellular domain of the wild-type class II alpha gene was fused via a GGGS linker to an acidic leucine zipper, followed by a GS linker to a 6xHis tag (see SEQ ID NO: 8). The extracellular domain of the class II beta gene carrying the mutation (see SEQ ID NO: 3) was similarly linked via a GGGS linker (see SEQ ID NO: 4) to a basic leucine zipper. HEK293T cells were transfected with the alpha and beta genes using a 293GPG cell-based retroviral system and cultured in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin. For DR1 dimer staining, HEK293T cells stably secreting soluble DR1 L114W / V143M+2reps , DR7 L114W / V143M+2reps , and DR11 L114W / V143M+2reps ​HEK293T cells expressing the protein (with S118H / T157I substitutions in addition to L114W / V143M (2 reps)) were grown to confluence and cultures were harvested after 48 hours. Supernatants containing soluble HLA class II were then mixed with 100 μg / ml of target peptide at 37°C for 20-24 hours to perform in vitro peptide exchange. Monomers that were not subjected to peptide exchange were used as controls. Concentrations of monomers were measured by specific ELISA using nickel-coated plates (XPressBio, Frederick, MD) and anti-His tag biotinylated mAb (AD1.1.10, R&D Systems, Minneapolis, MN). Soluble HLA class II monomers were dimerized at a 2:1 molar ratio using PE-conjugated anti-His mAb (AD1.1.10, Abeam, Cambridge, MA) for 1.5 hours at 4°C to perform staining.

[0223] HLA class II dimer staining

[0224] Primary T cells transduced with exogenous TCR genes were stained with HLA class II dimers at 37°C 46 were pre-treated with 50 nM dasatinib (LC Laboratories, Woburn, MA) for 30 minutes and stained with 5-15 μg / ml of class II dimers for 4-5 hours at room temperature. After washing, cells were counterstained with APC-Cy7-conjugated anti-CD4 mAb and PE-conjugated anti-Vβ22 mAb for cell surface molecules.

[0225] Protein modeling

[0226] HLA-DR1 and human CD4 complex model structures were predicted based on the structures of PDB IDs 3S5L and 3T0E using the Swiss-Model workspace for quaternary structure prediction.

[0227] Statistical analysis

[0228] Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA). Two-sample comparisons were performed using unpaired, two-tailed Student’s t test. No statistical methods were used to predetermine sample size. The investigator was not blinded to allocation during experiments or outcome assessment. Experiments were not randomized.

[0229] Bio-layer interferometry and steady-state analysis

[0230] The extracellular domain of human CD4 (residues 26-440 of NP_000607.1) followed by a GS linker and a 10x histidine (His) tag was stably expressed in the human cell line A375 (SEQ ID NOs: 11-12). Recombinant 10x His-tagged CD4 protein was purified from the supernatant using TALON metal affinity resin (TakaraBio, Shiga, Japan). The eluted protein was concentrated using an Amicon Ultra-15 spin column with a 10 kDa MWCO (MilliporeSigma, Burlington, MA). The buffer was exchanged into HBS-EP (GE Healthcare Life Sciences, Marlborough, MA) using a 10 kDa MWCO mini-dialyzer (Thermo Fisher Scientific, Waltham, MA). As confirmed by SDS-PAGE, the purity of the recombinant CD4 protein was always >90%.

[0231] Recombinant DR1 proteins consist of the extracellular domain of DRA1*01:01 and either wild-type DRB1*01:01 or the L114W / V143M+2 reps mutant. DRA1*01:01 is followed by an acidic leucine zipper, a GS linker, and a 10x histidine tag, while wild-type and mutant DRB1 are followed by a basic leucine zipper, a GS linker, and a biotinylation sequence (GLNDIFEAQKIEWHE; SEQ ID NO: 13). Both DRA and DRB genes were stably expressed in A375-BirA cells, which were transduced with a codon-optimized BirA gene encoding a 5' leader sequence and a 3' ER retention KDEL motif. Recombinant DR1 protein was purified from the supernatant using TALON metal affinity resin (Takara Bio, Shiga, Japan). Eluted protein was concentrated using Vivaspin 500 spin columns with a 10 kDa MWCO (GE Healthcare Life Sciences, Marlborough, MA) and reconstituted to working volume in PBS.

[0232] Wild-type DR1 and DR1 were measured by the Octet Red system (ForteBio, Fremont, CA). L114W / V143M+2repsBinding to CD4. Experiments were performed at 25 °C using 96-well OptiPlate (Perkin Elmer, Waltham, MA) with a sample volume of 200 μΐ and constant shaking at 1,000 rpm. Biotinylated recombinant DR1 was loaded onto a streptavidin-coated biosensor (ForteBio, Fremont, CA) until saturation, followed by baseline measurements in HBS-EP buffer. Association was measured by incubating the loaded sensor with titrated concentrations of recombinant CD4 (0.8125 to 26 μΜ) for 400 s, followed by dissociation in separate HBS-EP buffer for 300 s. Steady-state analysis was fitted using the one-site specific binding model in GraphPad Prism 7.0.

[0233] Table 3. Soluble 10x His-tagged CD4 nucleic acid sequences

[0234]

[0235]

[0236] Example 2 - DR molecules with enhanced CD4 binding capacity

[0237] Affinity-enhanced DR molecules were generated by introducing the L114W / V143M mutations to determine whether these substitutions could improve the binding of HLA-DR molecules, such as the DR1 allele (DRA1*01:01-DRB1*01:01), to CD4. In addition to positions 114 and 143, DRB1*01:01 encodes six different amino acids at positions 118, 139, 146, 157, 163, and 164 (Table 3). Figure 1F ) compared to DR1 L114W / V143M and wild-type DR1. DR1 L114W / V143M+6reps showed enhanced CD4 binding Figures 1G-1L and Figure 1M ) compared to DR1 L114W / V143M+6reps A library of DR1 L114W / V143M+2reps derived mutants showed reduced CD4 binding capacity, indicating that both the S118H and T157I mutations are critical Figures 1N-1O ).

[0238] In fact, DR1 L114W / V143M+6reps with the L114W / V143M+S118H / T157I substitutions (2 reps) in the beta chain had CD4 binding capacity comparable to DR1 Figures 2A-2C). These results indicate that the two additional substitutions at S118H and T157I are critical for the L114W / V143M mutation to improve DR1 binding to CD4.

[0239] In addition to positions 114 and 143, DR beta chains such as DRB1*03:01, 04:01, 07:01, 10:01, 11:01, and 13:01 also encode different amino acids at positions 118, 139, 146, 157, 163, and 164 ( Figure 2D ). Interestingly, comparing the CD4 binding activity between DR1 L114W / V143M+2reps and DR1 L114W / V143M+6reps mutants indicates that, unlike DR1, for DR3, DR4, DR7, DR10, DR11, and DR13, the L114W / V143M+2reps mutation is able to improve CD4 binding compared to the L114W / V143M+6reps mutation ( Figures 3A-3B ).

[0240] The affinity of wild-type DR1 and DR1 L114W / V143M+2reps mutants for CD4 was measured using a Biolayer Interferometry (BLI) binding assay. While no binding was detected between wild-type DR1 and CD4 ( Figure 3C ), DR1 L114W / V143M+2reps mutants bound CD4 with a KD of 14 μΜ ± 2.3 ( Figure 3D ).

[0241] Example 3 - Affinity matured DR dimers specificity and robustly stain cognate TCRs

[0242] The ability of affinity matured DR dimers carrying the mutations described in Example 2 to identify antigen-specific CD4 + T cells was assessed. DR1 L114W / V143M+2reps , DR7 L114W / V143M+2reps , and DR11 L114W / V143M+2reps dimers specifically stained DR1 -restricted TCR HA1.7 and SB95, DR7-restricted TCR SD334, and DR11 -restricted TCR F24, respectively ( Figures 5A-5L ). Complex staining of F24-transduced CD4 L114W / V143M+2reps T cells with anti-Vβ2 2mAb along with the corresponding DR11 + dimers confirmed that almost all TCR-transduced CD4 + T cells were successfully stained with the corresponding DR11 L114W / V143M+2reps dimers ( Figures 6A-6X ).

[0243] CD4 and DR1 L114W / V143M+2repsStructural models of the complex formed also show potential hydrophobic interactions of the L114W / V143M substitutions Figures 7A-7O ). In addition, hydrophobic packing was observed between P96 of the alpha-chain and S118H of the beta-chain Figures 7A-7O ), and the T157I substitution was found to be located in the beta-sheet surrounding V119, F112, 1127, V129, L147 and T157 ​ ). Additional mutations in the alpha and / or beta chain can further enhance the binding between class II and CD4. However, the use of soluble class II molecules with excessive CD4 binding capacity can lead to non-specific staining of T cells, with detrimental effects. + T cells, thereby having detrimental effects.

[0244] To identify affinity matured class II molecules, this example details multiple mutations in the beta chain, but not the alpha chain, as the beta chain has a more direct interaction with CD4 than the alpha chain. Additional mutations in the alpha and / or beta chain can further enhance the binding between class II and CD4. However, the use of soluble class II molecules with excessive CD4 binding capacity can lead to non-specific staining of T cells, with detrimental effects. + T cells, thereby having detrimental effects.

[0245] In summary, CD4 + T cells play a key role in the development of autoimmune diseases as well as in protection against pathogenic infections and cancer. The new HLA class II multimer technology described herein can better facilitate the study of HLA class II restricted CD4 + T cell responses to HLA-DR alleles.

[0246] Example 4

[0247] Memory CD4 L114W / V143M+2reps T cells were stained ex vivo with DR1 + dimer specific for the influenza virus hemagglutinin (Flu-HA) peptide, in the absence of in vitro stimulation. For Flu-HA 117-136 - and Flu-HA 306-318 , a small subset of CD4 + T cells was positively stained with DR1 L114W / V143M+2reps dimer( ​ ​ ). Wild type DR1, DR1 L114W / V143M and DR1 L114W / V143M+6reps dimer and DR1 L114W / V143M+2reps dimer were generated and compared for their staining of TCR transduced CD4 + T cells. Wild type DR1 and DR1 L114W / V143MDimer in CD4 + Very little cognate TCR (HA1.7) was detected on T cells, while DR1 L114W / V143M+2reps and DR1 L114W / V143M+6reps dimer showed similar robust staining. Importantly, DR1 L114W / V143M+2reps dimer stained HA1.7 transduced CD4 + T cells more robustly and isolated better than wild type DR1 dextramer ​ ​ ). To validate DR1 L114W / V143M+2reps dimer staining, dimer + CD4 + T cells were expanded in vitro in a peptide specific manner. Clonal DR1 restricted TCR genes specific for HSD17B12 225-244 and LY6K 99-118 were identified. When cloned and reconstituted in primary CD4 + T cells, both TCRs (Table 4) were successfully stained by cognate DR1 L114W / V143M+2reps dimer and functioned in a DR1 restricted and antigen specific manner ​ ​ ).

[0248] Table 4: TCR sequences

[0249]

[0250] Methods

[0251] Cells

[0252] Peripheral mononuclear cells were obtained by density gradient centrifugation. K562-based artificial antigen presenting cells (aAPC) expressing various HLA class II genes as single HLA alleles along with CD80 and CD83 have been previously reported (see Butler, M.O., et al., PLoS One 7, e30229 (2012)). The Jurkat 76 cell line is a T cell leukemia cell line that lacks endogenous TCR, CD4 and CD8 expression (see Heemskerk, M.H., et al., Blood 102, 3530-3540 (2003)). Jurkat 76 / CD4 cells were generated by retroviral transduction of the human CD4 gene. A375 cells are a melanoma cell line. HEK293T cells and A375 cells were grown in DMEM supplemented with 10% FBS and 50 pg / ml gentamicin. The Jurkat 76 cell line was cultured in RPMI 1640 supplemented with 10% FBS and 50 pg / ml gentamicin.

[0253] Peptides

[0254] Synthetic peptides were dissolved in DMSO at 50 mg / ml.

[0255] Genes

[0256] New TCR genes were cloned by 5'-rapid amplification of cDNA ends (RACE) PCR and sequenced as previously described (Nakatsugawa, M. et al. Sci Rep 6, 23821 (2016); Nakatsugawa, M. et al. J Immunol 194, 3487-3500 (2015); Ochi, T. et al. Cancer Immunol Res 3, 1070-1081 (2015)). All genes were cloned into pMX retroviral vectors and transduced into cell lines using a retroviral system based on 293GPG and PG13 cells (see Hirano, N. et al. Blood 107, 1528-1536 (2006); Butler, M. O. et al. Clin Cancer Res 13, 1857-1867 (2007); Hirano, N. et al. Clin Cancer Res 12, 2967-2975 (2006)).

[0257] Antibodies

[0258] The following antibodies were used for flow cytometry analysis: APC-Cy7-conjugated anti-CD4 (RPA-T4; see Wooldridge, L. et al. Eur J Immunol 36, 1847-1855 (2006)) and PE-conjugated anti-His tag (AD1.1.10, ABCAM, Cambridge, MA). Dead cells were distinguished with the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit. Stained cells were analyzed with a FACSCanto II or LSRFortessa X-20. Cell sorting was performed using a FACSAria II. Data analysis was performed using FlowJo software (version 9.9.6).

[0259] Generation of HLA class II monomers and dimers was performed using a 293GPG cell-based retroviral system to transfect HEK293T cells with a and b genes (see Hirano, N., et al., Blood 107, 1528-1536 (2006); Butler, M.O., et al., Clin Cancer Res 13, 1857-1867 (2007); Hirano, N., et al., Blood 108, 2662-2668 (2006)) and cultured in DMEM supplemented with 10% FBS and 50 pg / ml gentamicin. After 48 hours, the conditioned media was harvested. The supernatant containing soluble HLA class II was then mixed with 100 pg / ml of the peptide of interest at 37°C for 20-24 hours to perform in vitro peptide exchange. Monomer concentration was measured by specific ELISA using nickel-coated plates and biotinylated mAb against His tag. Soluble HLA class II monomers were dimerized using PE-conjugated anti-His mAb (AD1.1.10) at a 2:1 molar ratio at 4°C for 1.5 hours to perform staining.

[0260] DR1 -restricted antigen-specific CD4 + Stimulation of T cells

[0261] Purification of CD4 + T cells and then stimulated with DR1 -expressing aAPC pulsed with DR1 -restricted peptide at 10 pg / ml and irradiated at 200 Gy at an E:T ratio of 20:1. After 48 hours, 10 IU / ml IL-2 and 10 ng / ml IL-15 were added to the CD4 + T cells. Medium supplemented with IL-2 (10 IU / ml) and IL-15 (10 ng / ml) was replenished every 2-3 days. After two weeks, T cells were stained with DR1 L114W / V143M+2reps dimers.

[0262] HLA class II dimers and dextramer staining

[0263] Comparison of DR1 dextramer in multimer staining analysis. Primary CD4 + T cells transduced with antigen-specific TCR genes were pre-treated with 50 nM dasatinib for 30 minutes at 37°C and stained with 5-15 pg / ml class II dimers for 4-5 hours at room temperature. After washing, cells were counterstained with APC-Cy7-conjugated anti-CD4 mAb for cell surface molecules.

[0264] Unstimulated CD4 + T cells from melanoma patients dimer staining

[0265] One million CD4 + T cells were purified and pre-treated with 50 nM dasatinib for 30 min at 37°C. Cells were stained with 5-15 pg / ml class II dimers for 4-5 h at room temperature. After washing, cells were re-stained for cell surface molecules with APC-Cy7-conjugated anti-CD4 mAb. Dimers + Absolute counts of cells.

[0266] ELISPOT assay

[0267] Cytokine ELISPOT assays were performed as previously reported (see, e.g., Yamashita, Y. et al., Nat Commun 8, 15244 (2017); and Anczurowski, M. et al., Sci Rep 8, 4804 (2018)). SEQUENCE LISTING <110> University Health Network <120> MHC class II molecules and methods of use thereof <130> 4285.012PC02 / C-K / BMD <150> US 62 / 880,509 <151> 2019-07-30 <150> US 63 / 029,115 <151> 2020-05-22 <160> 24 <170> PatentIn version 3.5 <210> 1 <211> 198 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 1 Gly Asp Thr Arg Pro Arg Phe Leu Trp Gin Leu Lys Phe Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cys Ile Tyr 20 25 30 Asn Gin Glu Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Leu Leu Glu Gin Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gin Arg Arg Val Glu 85 90 95 Pro Lys Val Thr Val Tyr Pro Ser Lys Thr Gin Pro Leu Gin His His 100 105 110 Asn Leu Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu 115 120 125 Val Arg Trp Phe Arg Asn Gly Gin Glu Glu Lys Ala Gly Val Val Ser 130 135 140 Thr Gly Leu Ile Gin Asn Gly Asp Trp Thr Phe Gin Thr Leu Val Met 145 150 155 160 Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gin Val Glu 165 170 175 His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser 180 185 190 Glu Ser Ala Gln Ser Lys 195 <210> 2 <211> 594 <212> PRT <213> 人工序列(Artificial Sequence) <220> <223> 合成构建体 <400> 2 Gly Gly Gly Gly Ala Cys Ala Cys Cys Cys Gly Ala Cys Cys Ala Cys 1 5 10 15 Gly Thr Thr Thr Cys Thr Thr Gly Thr Gly Gly Cys Ala Gly Cys Thr 20 25 30 Thr Ala Ala Gly Thr Thr Thr Gly Ala Ala Thr Gly Thr Cys Ala Thr 35 40 45 Thr Thr Cys Thr Thr Cys Ala Ala Thr Gly Gly Gly Ala Cys Gly Gly 50 55 60 Ala Gly Cys Gly Gly Gly Thr Gly Cys Gly Gly Thr Thr Gly Cys Thr 65 70 75 80 Gly Gly Ala Ala Ala Gly Ala Thr Gly Cys Ala Thr Cys Thr Ala Thr 85 90 95 Ala Ala Cys Cys Ala Ala Gly Ala Gly Gly Ala Gly Thr Cys Cys Gly 100 105 110 Thr Gly Cys Gly Cys Thr Thr Cys Gly Ala Cys Ala Gly Cys Gly Ala 115 120 125 Cys Gly Thr Gly Gly Gly Gly Gly Ala Gly Thr Ala Cys Cys Gly Gly 130 135 140 Gly Cys Gly Gly Thr Gly Ala Cys Gly Gly Ala Gly Cys Thr Gly Gly 145 150 155 160 Gly Gly Cys Gly Gly Cys Cys Thr Gly Ala Thr Gly Cys Cys Gly Ala 165 170 175 Gly Thr Ala Cys Thr Gly Gly Ala Ala Cys Ala Gly Cys Cys Ala Gly 180 185 190 Ala Ala Gly Gly Ala Cys Cys Thr Cys Cys Thr Gly Gly Ala Gly Cys 195 200 205 Ala Gly Ala Gly Gly Cys Gly Gly Gly Cys Cys Gly Cys Gly Gly Thr 210 215 220 Gly Gly Ala Cys Ala Cys Cys Thr Ala Cys Thr Gly Cys Ala Gly Ala 225 230 235 240 Cys Ala Cys Ala Ala Cys Thr Ala Cys Gly Gly Gly Gly Thr Thr Gly 245 250 255 Gly Thr Gly Ala Gly Ala Gly Cys Thr Thr Cys Ala Cys Ala Gly Thr 260 265 270 Gly Cys Ala Gly Cys Gly Gly Cys Gly Ala Gly Thr Thr Gly Ala Gly 275 280 285 Cys Cys Thr Ala Ala Gly Gly Thr Gly Ala Cys Thr Gly Thr Gly Thr 290 295 300 Ala Thr Cys Cys Thr Thr Cys Ala Ala Ala Gly Ala Cys Cys Cys Ala 305 310 315 320 Gly Cys Cys Cys Cys Thr Gly Cys Ala Gly Cys Ala Cys Cys Ala Cys 325 330 335 Ala Ala Cys Cys Thr Cys Cys Thr Gly Gly Thr Cys Thr Gly Cys Thr 340 345 350 Cys Thr Gly Thr Gly Ala Gly Thr Gly Gly Thr Thr Thr Cys Thr Ala 355 360 365 Thr Cys Cys Ala Gly Gly Cys Ala Gly Cys Ala Thr Thr Gly Ala Ala 370 375 380 Gly Thr Cys Ala Gly Gly Thr Gly Gly Thr Thr Cys Cys Gly Gly Ala 385 390 395 400 Ala Cys Gly Gly Cys Cys Ala Gly Gly Ala Ala Gly Ala Gly Ala Ala 405 410 415 Gly Gly Cys Thr Gly Gly Gly Gly Thr Gly Gly Thr Gly Thr Cys Cys 420 425 430 Ala Cys Ala Gly Gly Cys Cys Thr Gly Ala Thr Cys Cys Ala Gly Ala 435 440 445 Ala Thr Gly Gly Ala Gly Ala Thr Thr Gly Gly Ala Cys Cys Thr Thr 450 455 460 Cys Cys Ala Gly Ala Cys Cys Cys Thr Gly Gly Thr Gly Ala Thr Gly 465 470 475 480 Cys Thr Gly Gly Ala Ala Ala Cys Ala Gly Thr Thr Cys Cys Thr Cys 485 490 495 Gly Gly Ala Gly Thr Gly Gly Ala Gly Ala Gly Gly Thr Thr Thr Ala 500 505 510 Cys Ala Cys Cys Thr Gly Cys Cys Ala Ala Gly Thr Gly Gly Ala Gly 515 520 525 Cys Ala Cys Cys Cys Ala Ala Gly Thr Gly Thr Gly Ala Cys Gly Ala 530 535 540 Gly Cys Cys Cys Thr Cys Thr Cys Ala Cys Ala Gly Thr Gly Gly Ala 545 550 555 560 Ala Thr Gly Gly Ala Gly Ala Gly Cys Ala Cys Gly Gly Thr Cys Thr 565 570 575 Gly Ala Ala Thr Cys Thr Gly Cys Ala Cys Ala Gly Ala Gly Cys Ala 580 585 590 Ala Gly <210> 3 <211> 198 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 3 Gly Asp Thr Arg Pro Arg Phe Leu Trp Gin Leu Lys Phe Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cys Ile Tyr 20 25 30 Asn Gin Glu Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin 50 55 60 Lys Asp Leu Leu Glu Gin Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gin Arg Arg Val Glu 85 90 95 Pro Lys Val Thr Val Tyr Pro Ser Lys Thr Gin Pro Leu Gin His His 100 105 110 Asn Trp Leu Val Cys His Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu 115 120 125 Val Arg Trp Phe Arg Asn Gly Gin Glu Glu Lys Ala Gly Val Met Ser 130 135 140 Thr Gly Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Ile Leu Val Met 145 150 155 160 Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu 165 170 175 His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser 180 185 190 Glu Ser Ala Gln Ser Lys 195 <210> 4 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 4 Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu 1 5 10 15 Ala Gly Asp Thr Arg Pro Arg Phe Leu Trp Gln Leu Lys Phe Glu Cys 20 25 30 His Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cys Ile 35 40 45 Tyr Asn Gln Glu Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 50 55 60 Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 65 70 75 80 Gln Lys Asp Leu Leu Glu Gin Arg Arg Ala Ala Val Asp Thr Tyr Cys 85 90 95 Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gin Arg Arg Val 100 105 110 Glu Pro Lys Val Thr Val Tyr Pro Ser Lys Thr Gin Pro Leu Gin His 115 120 125 His Asn Trp Leu Val Cys His Val Ser Gly Phe Tyr Pro Gly Ser Ile 130 135 140 Glu Val Arg Trp Phe Arg Asn Gly Gin Glu Glu Lys Ala Gly Val Met 145 150 155 160 Ser Thr Gly Leu Ile Gin Asn Gly Asp Trp Thr Phe Gin Ile Leu Val 165 170 175 Met Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gin Val 180 185 190 Glu His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg 195 200 205 Ser Glu Ser Ala Gin Ser Lys Gly Gly Gly Gly Ser Leu Glu Ile Glu 210 215 220 Ala Ala Phe Leu Glu Arg Glu Asn Thr Ala Leu Glu Thr Arg Val Ala 225 230 235 240 Glu Leu Arg Gin Arg Val Gin Arg Leu Arg Asn Arg Val Ser Gin Tyr 245 250 255 Arg Thr Arg Tyr Gly Pro Leu Gly Gly Gly Lys 260 265 <210> 5 <211> 266 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Synthetic construct <400> 5 Met Val Cys Leu Lys Leu Pro Gly Gly Ser Cys Met Thr Ala Leu Thr 1 5 10 15 Val Thr Leu Met Val Leu Ser Ser Pro Leu Ala Leu Ala Gly Asp Thr 20 25 30 Arg Pro Arg Phe Leu Trp Gin Leu Lys Phe Glu Cys His Phe Phe Asn 35 40 45 Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cys Ile Tyr Asn Gin Glu 50 55 60 Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr 65 70 75 80 Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu 85 90 95 Leu Glu Gin Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr 100 105 110 Gly Val Gly Glu Ser Phe Thr Val Gin Arg Arg Val Glu Pro Lys Val 115 120 125 Thr Val Tyr Pro Ser Lys Thr Gin Pro Leu Gin His His Asn Leu Leu 130 135 140 Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser He Glu Val Arg Trp 145 150 155 160 Phe Arg Asn Gly Gin Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu 165 170 175 He Gin Asn Gly Asp Trp Thr Phe Gin Thr Leu Val Met Leu Glu Thr 180 185 190 Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gin Val Glu His Pro Ser 195 200 205 Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala 210 215 220 Gln Ser Lys Met Leu Ser Gly Val Gly Gly Phe Val Leu Gly Leu Leu 225 230 235 240 Phe Leu Gly Ala Gly Leu Phe Ile Tyr Phe Arg Asn Gin Lys Gly His 245 250 255 Ser Gly Leu Gin Pro Thr Gly Phe Leu Ser 260 265 <210> 6 <211> 192 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Synthetic construct <400> 6 Ile Lys Glu Glu His Val lie lie Gin Ala Glu Phe Tyr Leu Asn Pro 1 5 10 15 Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu lie Phe 20 25 30 His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe 35 40 45 Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn lie Ala 50 55 60 Val Asp Lys Ala Asn Leu Glu lie Met Thr Lys Arg Ser Asn Tyr Thr 65 70 75 80 Pro lie Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser Pro 85 90 95 Val Glu Leu Arg Glu Pro Asn Val Leu lie Cys Phe lie Asp Lys Phe 100 105 110 Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro Val 115 120 125 Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His Leu 130 135 140 Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp Val 145 150 155 160 Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu Lys 165 170 175 His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu Asn 180 185 190 <210> 7 <211> 576 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 7 Ala Thr Cys Ala Ala Ala Gly Ala Ala Gly Ala Ala Cys Ala Thr Gly 1 5 10 15 Thr Gly Ala Thr Cys Ala Thr Cys Cys Ala Gly Gly Cys Cys Gly Ala 20 25 30 Gly Thr Thr Cys Thr Ala Thr Cys Thr Gly Ala Ala Thr Cys Cys Thr 35 40 45 Gly Ala Cys Cys Ala Ala Thr Cys Ala Gly Gly Cys Gly Ala Gly Thr 50 55 60 Thr Thr Ala Thr Gly Thr Thr Thr Gly Ala Cys Thr Thr Thr Gly Ala 65 70 75 80 Thr Gly Gly Thr Gly Ala Thr Gly Ala Gly Ala Thr Thr Thr Thr Cys 85 90 95 Cys Ala Thr Gly Thr Gly Gly Ala Thr Ala Thr Gly Gly Cys Ala Ala 100 105 110 Ala Gly Ala Ala Gly Gly Ala Gly Ala Cys Gly Gly Thr Cys Thr Gly 115 120 125 Gly Cys Gly Gly Cys Thr Thr Gly Ala Ala Gly Ala Ala Thr Thr Thr 130 135 140 Gly Gly Ala Cys Gly Ala Thr Thr Thr Gly Cys Cys Ala Gly Cys Thr 145 150 155 160 Thr Thr Gly Ala Gly Gly Cys Thr Cys Ala Ala Gly Gly Thr Gly Cys 165 170 175 Ala Thr Thr Gly Gly Cys Cys Ala Ala Cys Ala Thr Ala Gly Cys Thr 180 185 190 Gly Thr Gly Gly Ala Cys Ala Ala Ala Gly Cys Cys Ala Ala Cys Cys 195 200 205 Thr Gly Gly Ala Ala Ala Thr Cys Ala Thr Gly Ala Cys Ala Ala Ala 210 215 220 Gly Cys Gly Cys Thr Cys Cys Ala Ala Cys Thr Ala Thr Ala Cys Thr 225 230 235 240 Cys Cys Gly Ala Thr Cys Ala Cys Cys Ala Ala Thr Gly Thr Ala Cys 245 250 255 Cys Thr Cys Cys Ala Gly Ala Gly Gly Thr Ala Ala Cys Thr Gly Thr 260 265 270 Gly Cys Thr Cys Ala Cys Ala Ala Ala Cys Ala Gly Cys Cys Cys Thr 275 280 285 Gly Thr Gly Gly Ala Ala Cys Thr Gly Ala Gly Ala Gly Ala Gly Cys 290 295 300 Cys Cys Ala Ala Cys Gly Thr Cys Cys Thr Cys Ala Thr Cys Thr Gly 305 310 315 320 Thr Thr Thr Cys Ala Thr Ala Gly Ala Cys Ala Ala Gly Thr Thr Cys 325 330 335 Ala Cys Cys Cys Cys Ala Cys Cys Ala Gly Thr Gly Gly Thr Cys Ala 340 345 350 Ala Thr Gly Thr Cys Ala Cys Gly Thr Gly Gly Cys Thr Thr Cys Gly 355 360 365 Ala Ala Ala Thr Gly Gly Ala Ala Ala Ala Cys Cys Thr Gly Thr Cys 370 375 380 Ala Cys Cys Ala Cys Ala Gly Gly Ala Gly Thr Gly Thr Cys Ala Gly 385 390 395 400 Ala Gly Ala Cys Ala Gly Thr Cys Thr Thr Cys Cys Thr Gly Cys Cys 405 410 415 Cys Ala Gly Gly Gly Ala Ala Gly Ala Cys Cys Ala Cys Cys Thr Thr 420 425 430 Thr Thr Cys Cys Gly Cys Ala Ala Gly Thr Thr Cys Cys Ala Cys Thr 435 440 445 Ala Thr Cys Thr Cys Cys Cys Cys Thr Thr Cys Cys Thr Gly Cys Cys 450 455 460 Cys Thr Cys Ala Ala Cys Thr Gly Ala Gly Gly Ala Cys Gly Thr Thr 465 470 475 480 Thr Ala Cys Gly Ala Cys Thr Gly Cys Ala Gly Gly Gly Thr Gly Gly 485 490 495 Ala Gly Cys Ala Cys Thr Gly Gly Gly Gly Cys Thr Thr Gly Gly Ala 500 505 510 Thr Gly Ala Gly Cys Cys Thr Cys Thr Thr Cys Thr Cys Ala Ala Gly 515 520 525 Cys Ala Cys Thr Gly Gly Gly Ala Gly Thr Thr Thr Gly Ala Thr Gly 530 535 540 Cys Thr Cys Cys Ala Ala Gly Cys Cys Cys Thr Cys Thr Cys Cys Cys 545 550 555 560 Ala Gly Ala Gly Ala Cys Thr Ala Cys Ala Gly Ala Gly Ala Ala Cys 565 570 575 <210> 8 <211> 269 <212> PRT <213> 人工序列(Artificial Sequence) <220> <223> 合成构建体 <400> 8 Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu 1 5 10 15 Ala Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn 20 25 30 Pro Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile 35 40 45 Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu 50 55 60 Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn lie 65 70 75 80 Ala Val Asp Lys Ala Asn Leu Glu lie Met Thr Lys Arg Ser Asn Tyr 85 90 95 Thr Pro lie Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser 100 105 110 Pro Val Glu Leu Arg Glu Pro Asn Val Leu lie Cys Phe lie Asp Lys 115 120 125 Phe Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro 130 135 140 Val Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His 145 150 155 160 Leu Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp 165 170 175 Val Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu 180 185 190 Lys His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu 195 200 205 Asn Gly Gly Gly Gly Ser Leu Glu lie Arg Ala Ala Phe Leu Arg Gin 210 215 220 Arg Asn Thr Ala Leu Arg Thr Glu Val Ala Glu Leu Glu Gin Glu Val 225 230 235 240 Gln Arg Leu Glu Asn Glu Val Ser Gin Tyr Glu Thr Arg Tyr Gly Pro 245 250 255 Leu Gly Gly Gly Lys Gly Ser His His His His His His 260 265 <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 9 Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu 1 5 10 15 Ala <210> 10 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 10 Met Asn Arg Gly Val Pro Phe Arg His Leu Leu Leu Val Leu Gin Leu 1 5 10 15 Ala Leu Leu Pro Ala Ala Thr Gin Gly Lys Lys Val Val Leu Gly Lys 20 25 30 Lys Gly Asp Thr Val Glu Leu Thr Cys Thr Ala Ser Gin Lys Lys Ser 35 40 45 Ile Gln Phe His Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn 50 55 60 Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala 65 70 75 80 Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile 85 90 95 Lys Asn Leu Lys Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu 100 105 110 Asp Gln Lys Glu Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn 115 120 125 Ser Asp Thr His Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu 130 135 140 Ser Pro Pro Gly Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly 145 150 155 160 Lys Asn Ile Gln Gly Gly Lys Thr Leu Ser Val Ser Gln Leu Glu Leu 165 170 175 Gln Asp Ser Gly Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys 180 185 190 Val Glu Phe Lys Ile Asp Ile Val Val Leu Ala Phe Gln Lys Ala Ser 195 200 205 Ser Ile Val Tyr Lys Lys Glu Gly Glu Gin Val Glu Phe Ser Phe Pro 210 215 220 Leu Ala Phe Thr Val Glu Lys Leu Thr Gly Ser Gly Glu Leu Trp Trp 225 230 235 240 Gln Ala Glu Arg Ala Ser Ser Ser Lys Ser Trp Ile Thr Phe Asp Leu 245 250 255 Lys Asn Lys Glu Val Ser Val Lys Arg Val Thr Gin Asp Pro Lys Leu 260 265 270 Gln Met Gly Lys Lys Leu Pro Leu His Leu Thr Leu Pro Gin Ala Leu 275 280 285 Pro Gin Tyr Ala Gly Ser Gly Asn Leu Thr Leu Ala Leu Glu Ala Lys 290 295 300 Thr Gly Lys Leu His Gin Glu Val Asn Leu Val Val Met Arg Ala Thr 305 310 315 320 Gln Leu Gin Lys Asn Leu Thr Cys Glu Val Trp Gly Pro Thr Ser Pro 325 330 335 Lys Leu Met Leu Ser Leu Lys Leu Glu Asn Lys Glu Ala Lys Val Ser 340 345 350 Lys Arg Glu Lys Ala Val Trp Val Leu Asn Pro Glu Ala Gly Met Trp 355 360 365 Gln Cys Leu Leu Ser Asp Ser Gly Gln Val Leu Leu Glu Ser Asn Ile 370 375 380 Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Met Ala Leu Ile 385 390 395 400 Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile 405 410 415 Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met 420 425 430 Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro 435 440 445 His Arg Phe Gln Lys Thr Cys Ser Pro Ile 450 455 <210> 11 <211> 198 <212> PRT <213> 人工序列(Artificial Sequence) <220> <223> 合成构建体 <400> 11 Gly Asp Thr Arg Pro Arg Phe Leu Trp Gln Leu Lys Phe Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cys Ile Tyr 20 25 30 Asn Gln Glu Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Leu Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val Glu 85 90 95 Pro Lys Val Thr Val Tyr Pro Ser Lys Thr Gln Pro Leu Gln His His 100 105 110 Asn Trp Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu 115 120 125 Val Arg Trp Phe Arg Asn Gly Gln Glu Glu Lys Ala Gly Val Met Ser 130 135 140 Thr Gly Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met 145 150 155 160 Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu 165 170 175 His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser 180 185 190 Glu Ser Ala Gln Ser Lys 195 <210> 12 <211> 198 <212> PRT <213> 人工序列(Artificial Sequence) <220> <223> 合成构建体 <400> 12 Gly Asp Thr Arg Pro Arg Phe Leu Trp Gln Leu Lys Phe Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cys Ile Tyr 20 25 30 Asn Gln Glu Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Leu Leu Glu Gln Arg Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val Glu 85 90 95 Pro Lys Val Thr Val Tyr Pro Ser Lys Thr Gln Pro Leu Gln His His 100 105 110 Asn Trp Leu Val Cys His Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu 115 120 125 Val Arg Trp Phe Arg Asn Gly Gln Glu Glu Thr Ala Gly Val Met Ser 130 135 140 Thr Asn Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Ile Leu Val Met 145 150 155 160 Leu Glu Met Thr Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu 165 170 175 His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser 180 185 190 Glu Ser Ala Gln Ser Lys 195 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 13 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 1 5 10 15 <210> 14 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 14 Met Met Arg Pro lie Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu 1 5 10 15 Ala Gly Asp Thr Arg Pro Arg Phe Leu Trp Gin Leu Lys Phe Glu Cys 20 25 30 His Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cys lie 35 40 45 Tyr Asn Gin Glu Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 50 55 60 Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 65 70 75 80 Gln Lys Asp Leu Leu Glu Gin Arg Arg Ala Ala Val Asp Thr Tyr Cys 85 90 95 Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gin Arg Arg Val 100 105 110 Glu Pro Lys Val Thr Val Tyr Pro Ser Lys Thr Gin Pro Leu Gin His 115 120 125 His Asn Trp Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser lie 130 135 140 Glu Val Arg Trp Phe Arg Asn Gly Gin Glu Glu Lys Ala Gly Val Met 145 150 155 160 Ser Thr Gly Leu lie Gin Asn Gly Asp Trp Thr Phe Gin Thr Leu Val 165 170 175 Met Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gin Val 180 185 190 Glu His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg 195 200 205 Ser Glu Ser Ala Gin Ser Lys Gly Gly Gly Gly Ser Leu Glu lie Gin 210 215 220 Ala Ala Phe Leu Glu Arg Glu Asn Thr Ala Leu Glu Thr Arg Val Ala 225 230 235 240 Glu Leu Arg Gin Arg Val Gin Arg Leu Arg Asn Arg Val Ser Gin Tyr 245 250 255 Arg Thr Arg Tyr Gly Pro Leu Gly Gly Gly Lys 260 265 <210> 15 <211> 284 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 15 Met Met Arg Pro lie Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu 1 5 10 15 Ala Gly Asp Thr Arg Pro Arg Phe Leu Trp Gin Leu Lys Phe Glu Cys 20 25 30 His Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cys Ile 35 40 45 Tyr Asn Gin Glu Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 50 55 60 Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 65 70 75 80 Gln Lys Asp Leu Leu Glu Gin Arg Arg Ala Ala Val Asp Thr Tyr Cys 85 90 95 Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gin Arg Arg Val 100 105 110 Glu Pro Lys Val Thr Val Tyr Pro Ser Lys Thr Gin Pro Leu Gin His 115 120 125 His Asn Leu Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile 130 135 140 Glu Val Arg Trp Phe Arg Asn Gly Gin Glu Glu Lys Ala Gly Val Val 145 150 155 160 Ser Thr Gly Leu Ile Gin Asn Gly Asp Trp Thr Phe Gin Thr Leu Val 165 170 175 Met Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val 180 185 190 Glu His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg 195 200 205 Ser Glu Ser Ala Gln Ser Lys Gly Gly Gly Gly Ser Leu Glu Ile Glu 210 215 220 Ala Ala Phe Leu Glu Arg Glu Asn Thr Ala Leu Glu Thr Arg Val Ala 225 230 235 240 Glu Leu Arg Gln Arg Val Gln Arg Leu Arg Asn Arg Val Ser Gln Tyr 245 250 255 Arg Thr Arg Tyr Gly Pro Leu Gly Gly Gly Lys Gly Ser Gly Leu Asn 260 265 270 Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 275 280 <210> 16 <211> 855 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 16 Ala Thr Gly Ala Thr Gly Cys Gly Gly Cys Cys Cys Ala Thr Cys Gly 1 5 10 15 Thr Gly Cys Thr Gly Gly Thr Gly Cys Thr Gly Cys Thr Gly Thr Thr 20 25 30 Cys Gly Cys Cys Ala Cys Ala Thr Cys Thr Gly Cys Cys Cys Thr Gly 35 40 45 Gly Cys Cys Gly Gly Gly Gly Ala Cys Ala Cys Cys Cys Gly Ala Cys 50 55 60 Cys Ala Cys Gly Thr Thr Thr Cys Thr Thr Gly Thr Gly Gly Cys Ala 65 70 75 80 Gly Cys Thr Thr Ala Ala Gly Thr Thr Thr Gly Ala Ala Thr Gly Thr 85 90 95 Cys Ala Thr Thr Thr Cys Thr Thr Cys Ala Ala Thr Gly Gly Gly Ala 100 105 110 Cys Gly Gly Ala Gly Cys Gly Gly Gly Thr Gly Cys Gly Gly Thr Thr 115 120 125 Gly Cys Thr Gly Gly Ala Ala Ala Gly Ala Thr Gly Cys Ala Thr Cys 130 135 140 Thr Ala Thr Ala Ala Cys Cys Ala Ala Gly Ala Gly Gly Ala Gly Thr 145 150 155 160 Cys Cys Gly Thr Gly Cys Gly Cys Thr Thr Cys Gly Ala Cys Ala Gly 165 170 175 Cys Gly Ala Cys Gly Thr Gly Gly Gly Gly Gly Ala Gly Thr Ala Cys 180 185 190 Cys Gly Gly Gly Cys Gly Gly Thr Gly Ala Cys Gly Gly Ala Gly Cys 195 200 205 Thr Gly Gly Gly Gly Cys Gly Gly Cys Cys Thr Gly Ala Thr Gly Cys 210 215 220 Cys Gly Ala Gly Thr Ala Cys Thr Gly Gly Ala Ala Cys Ala Gly Cys 225 230 235 240 Cys Ala Gly Ala Ala Gly Gly Ala Cys Cys Thr Cys Cys Thr Gly Gly 245 250 255 Ala Gly Cys Ala Gly Ala Gly Gly Cys Gly Gly Gly Cys Cys Gly Cys 260 265 270 Gly Gly Thr Gly Gly Ala Cys Ala Cys Cys Thr Ala Cys Thr Gly Cys 275 280 285 Ala Gly Ala Cys Ala Cys Ala Ala Cys Thr Ala Cys Gly Gly Gly Gly 290 295 300 Thr Thr Gly Gly Thr Gly Ala Gly Ala Gly Cys Thr Thr Cys Ala Cys 305 310 315 320 Ala Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Gly Ala Gly Thr Thr 325 330 335 Gly Ala Gly Cys Cys Thr Ala Ala Gly Gly Thr Gly Ala Cys Thr Gly 340 345 350 Thr Gly Thr Ala Thr Cys Cys Thr Thr Cys Ala Ala Ala Gly Ala Cys 355 360 365 Cys Cys Ala Gly Cys Cys Cys Cys Thr Gly Cys Ala Gly Cys Ala Cys 370 375 380 Cys Ala Cys Ala Ala Cys Cys Thr Cys Cys Thr Gly Gly Thr Cys Thr 385 390 395 400 Gly Cys Thr Cys Thr Gly Thr Gly Ala Gly Thr Gly Gly Thr Thr Thr 405 410 415 Cys Thr Ala Thr Cys Cys Ala Gly Gly Cys Ala Gly Cys Ala Thr Thr 420 425 430 Gly Ala Ala Gly Thr Cys Ala Gly Gly Thr Gly Gly Thr Thr Cys Cys 435 440 445 Gly Gly Ala Ala Cys Gly Gly Cys Cys Ala Gly Gly Ala Ala Gly Ala 450 455 460 Gly Ala Ala Gly Gly Cys Thr Gly Gly Gly Gly Thr Gly Gly Thr Gly 465 470 475 480 Thr Cys Cys Ala Cys Ala Gly Gly Cys Cys Thr Gly Ala Thr Cys Cys 485 490 495 Ala Gly Ala Ala Thr Gly Gly Ala Gly Ala Thr Thr Gly Gly Ala Cys 500 505 510 Cys Thr Thr Cys Cys Ala Gly Ala Cys Cys Cys Thr Gly Gly Thr Gly 515 520 525 Ala Thr Gly Cys Thr Gly Gly Ala Ala Ala Cys Ala Gly Thr Thr Cys 530 535 540 Cys Thr Cys Gly Gly Ala Gly Thr Gly Gly Ala Gly Ala Gly Gly Thr 545 550 555 560 Thr Thr Ala Cys Ala Cys Cys Thr Gly Cys Cys Ala Ala Gly Thr Gly 565 570 575 Gly Ala Gly Cys Ala Cys Cys Cys Ala Ala Gly Thr Gly Thr Gly Ala 580 585 590 Cys Gly Ala Gly Cys Cys Cys Thr Cys Thr Cys Ala Cys Ala Gly Thr 595 600 605 Gly Gly Ala Ala Thr Gly Gly Ala Gly Ala Gly Cys Ala Cys Gly Gly 610 615 620 Thr Cys Thr Gly Ala Ala Thr Cys Thr Gly Cys Ala Cys Ala Gly Ala 625 630 635 640 Gly Cys Ala Ala Gly Gly Gly Cys Gly Gly Cys Gly Gly Ala Gly Gly 645 650 655 Cys Ala Gly Cys Cys Thr Gly Gly Ala Ala Ala Thr Cys Gly Ala Gly 660 665 670 Gly Cys Cys Gly Cys Cys Thr Thr Cys Cys Thr Gly Gly Ala Ala Ala 675 680 685 Gly Ala Gly Ala Gly Ala Ala Cys Ala Cys Cys Gly Cys Cys Cys Thr 690 695 700 Gly Gly Ala Ala Ala Cys Cys Cys Gly Gly Gly Thr Gly Gly Cys Cys 705 710 715 720 Gly Ala Gly Cys Thr Gly Ala Gly Ala Cys Ala Gly Ala Gly Ala Gly 725 730 735 Thr Gly Cys Ala Gly Ala Gly Ala Cys Thr Gly Cys Gly Gly Ala Ala 740 745 750 Cys Cys Gly Gly Gly Thr Gly Thr Cys Cys Cys Ala Gly Thr Ala Cys 755 760 765 Cys Gly Gly Ala Cys Cys Ala Gly Ala Thr Ala Thr Gly Gly Cys Cys 770 775 780 Cys Thr Cys Thr Gly Gly Gly Ala Gly Gly Cys Gly Gly Cys Ala Ala 785 790 795 800 Ala Gly Gly Gly Thr Cys Cys Gly Gly Cys Thr Thr Gly Ala Ala Cys 805 810 815 Gly Ala Cys Ala Thr Thr Thr Thr Thr Gly Ala Gly Gly Cys Cys Cys 820 825 830 Ala Gly Ala Ala Gly Ala Thr Ala Gly Ala Gly Thr Gly Gly Cys Ala 835 840 845 Cys Gly Ala Gly Thr Gly Ala 850 855 <210> 17 <211> 284 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 17 Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu 1 5 10 15 Ala Gly Asp Thr Arg Pro Arg Phe Leu Trp Gin Leu Lys Phe Glu Cys 20 25 30 His Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu Glu Arg Cys Ile 35 40 45 Tyr Asn Gin Glu Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 50 55 60 Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 65 70 75 80 Gln Lys Asp Leu Leu Glu Gin Arg Arg Ala Ala Val Asp Thr Tyr Cys 85 90 95 Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gin Arg Arg Val 100 105 110 Glu Pro Lys Val Thr Val Tyr Pro Ser Lys Thr Gin Pro Leu Gin His 115 120 125 His Asn Trp Leu Val Cys His Val Ser Gly Phe Tyr Pro Gly Ser Ile 130 135 140 Glu Val Arg Trp Phe Arg Asn Gly Gin Glu Glu Lys Ala Gly Val Met 145 150 155 160 Ser Thr Gly Leu Ile Gin Asn Gly Asp Trp Thr Phe Gin Ile Leu Val 165 170 175 Met Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gin Val 180 185 190 Glu His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg 195 200 205 Ser Glu Ser Ala Gin Ser Lys Gly Gly Gly Gly Ser Leu Glu Ile Glu 210 215 220 Ala Ala Phe Leu Glu Arg Glu Asn Thr Ala Leu Glu Thr Arg Val Ala 225 230 235 240 Glu Leu Arg Gln Arg Val Gln Arg Leu Arg Asn Arg Val Ser Gln Tyr 245 250 255 Arg Thr Arg Tyr Gly Pro Leu Gly Gly Gly Lys Gly Ser Gly Leu Asn 260 265 270 Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 275 280 <210> 18 <211> 855 <212> PRT <213> 人工序列(Artificial Sequence) <220> <223> 合成构建体 <400> 18 Ala Thr Gly Ala Thr Gly Cys Gly Gly Cys Cys Cys Ala Thr Cys Gly 1 5 10 15 Thr Gly Cys Thr Gly Gly Thr Gly Cys Thr Gly Cys Thr Gly Thr Thr 20 25 30 Cys Gly Cys Cys Ala Cys Ala Thr Cys Thr Gly Cys Cys Cys Thr Gly 35 40 45 Gly Cys Cys Gly Gly Gly Gly Ala Cys Ala Cys Cys Cys Gly Ala Cys 50 55 60 Cys Ala Cys Gly Thr Thr Thr Cys Thr Thr Gly Thr Gly Gly Cys Ala 65 70 75 80 Gly Cys Thr Thr Ala Ala Gly Thr Thr Thr Gly Ala Ala Thr Gly Thr 85 90 95 Cys Ala Thr Thr Thr Cys Thr Thr Cys Ala Ala Thr Gly Gly Gly Ala 100 105 110 Cys Gly Gly Ala Gly Cys Gly Gly Gly Thr Gly Cys Gly Gly Thr Thr 115 120 125 Gly Cys Thr Gly Gly Ala Ala Ala Gly Ala Thr Gly Cys Ala Thr Cys 130 135 140 Thr Ala Thr Ala Ala Cys Cys Ala Ala Gly Ala Gly Gly Ala Gly Thr 145 150 155 160 Cys Cys Gly Thr Gly Cys Gly Cys Thr Thr Cys Gly Ala Cys Ala Gly 165 170 175 Cys Gly Ala Cys Gly Thr Gly Gly Gly Gly Gly Ala Gly Thr Ala Cys 180 185 190 Cys Gly Gly Gly Cys Gly Gly Thr Gly Ala Cys Gly Gly Ala Gly Cys 195 200 205 Thr Gly Gly Gly Gly Cys Gly Gly Cys Cys Thr Gly Ala Thr Gly Cys 210 215 220 Cys Gly Ala Gly Thr Ala Cys Thr Gly Gly Ala Ala Cys Ala Gly Cys 225 230 235 240 Cys Ala Gly Ala Ala Gly Gly Ala Cys Cys Thr Cys Cys Thr Gly Gly 245 250 255 Ala Gly Cys Ala Gly Ala Gly Gly Cys Gly Gly Gly Cys Cys Gly Cys 260 265 270 Gly Gly Thr Gly Gly Ala Cys Ala Cys Cys Thr Ala Cys Thr Gly Cys 275 280 285 Ala Gly Ala Cys Ala Cys Ala Ala Cys Thr Ala Cys Gly Gly Gly Gly 290 295 300 Thr Thr Gly Gly Thr Gly Ala Gly Ala Gly Cys Thr Thr Cys Ala Cys 305 310 315 320 Ala Gly Thr Gly Cys Ala Gly Cys Gly Gly Cys Gly Ala Gly Thr Thr 325 330 335 Gly Ala Gly Cys Cys Thr Ala Ala Gly Gly Thr Gly Ala Cys Thr Gly 340 345 350 Thr Gly Thr Ala Thr Cys Cys Thr Thr Cys Ala Ala Ala Gly Ala Cys 355 360 365 Cys Cys Ala Gly Cys Cys Cys Cys Thr Gly Cys Ala Gly Cys Ala Cys 370 375 380 Cys Ala Cys Ala Ala Cys Thr Gly Gly Cys Thr Gly Gly Thr Cys Thr 385 390 395 400 Gly Cys Cys Ala Thr Gly Thr Gly Ala Gly Thr Gly Gly Thr Thr Thr 405 410 415 Cys Thr Ala Thr Cys Cys Ala Gly Gly Cys Ala Gly Cys Ala Thr Thr 420 425 430 Gly Ala Ala Gly Thr Cys Ala Gly Gly Thr Gly Gly Thr Thr Cys Cys 435 440 445 Gly Gly Ala Ala Cys Gly Gly Cys Cys Ala Gly Gly Ala Ala Gly Ala 450 455 460 Gly Ala Ala Gly Gly Cys Thr Gly Gly Gly Gly Thr Gly Ala Thr Gly 465 470 475 480 Thr Cys Cys Ala Cys Ala Gly Gly Cys Cys Thr Gly Ala Thr Cys Cys 485 490 495 Ala Gly Ala Ala Thr Gly Gly Ala Gly Ala Thr Thr Gly Gly Ala Cys 500 505 510 Cys Thr Thr Cys Cys Ala Gly Ala Thr Cys Cys Thr Gly Gly Thr Gly 515 520 525 Ala Thr Gly Cys Thr Gly Gly Ala Ala Ala Cys Ala Gly Thr Thr Cys 530 535 540 Cys Thr Cys Gly Gly Ala Gly Thr Gly Gly Ala Gly Ala Gly Gly Thr 545 550 555 560 Thr Thr Ala Cys Ala Cys Cys Thr Gly Cys Cys Ala Ala Gly Thr Gly 565 570 575 Gly Ala Gly Cys Ala Cys Cys Cys Ala Ala Gly Thr Gly Thr Gly Ala 580 585 590 Cys Gly Ala Gly Cys Cys Cys Thr Cys Thr Cys Ala Cys Ala Gly Thr 595 600 605 Gly Gly Ala Ala Thr Gly Gly Ala Gly Ala Gly Cys Ala Cys Gly Gly 610 615 620 Thr Cys Thr Gly Ala Ala Thr Cys Thr Gly Cys Ala Cys Ala Gly Ala 625 630 635 640 Gly Cys Ala Ala Gly Gly Gly Cys Gly Gly Cys Gly Gly Ala Gly Gly 645 650 655 Cys Ala Gly Cys Cys Thr Gly Gly Ala Ala Ala Thr Cys Gly Ala Gly 660 665 670 Gly Cys Cys Gly Cys Cys Thr Thr Cys Cys Thr Gly Gly Ala Ala Ala 675 680 685 Gly Ala Gly Ala Gly Ala Ala Cys Ala Cys Cys Gly Cys Cys Cys Thr 690 695 700 Gly Gly Ala Ala Ala Cys Cys Cys Gly Gly Gly Thr Gly Gly Cys Cys 705 710 715 720 Gly Ala Gly Cys Thr Gly Ala Gly Ala Cys Ala Gly Ala Gly Ala Gly 725 730 735 Thr Gly Cys Ala Gly Ala Gly Ala Cys Thr Gly Cys Gly Gly Ala Ala 740 745 750 Cys Cys Gly Gly Gly Thr Gly Thr Cys Cys Cys Ala Gly Thr Ala Cys 755 760 765 Cys Gly Gly Ala Cys Cys Ala Gly Ala Thr Ala Thr Gly Gly Cys Cys 770 775 780 Cys Thr Cys Thr Gly Gly Gly Ala Gly Gly Cys Gly Gly Cys Ala Ala 785 790 795 800 Ala Gly Gly Gly Thr Cys Cys Gly Gly Cys Thr Thr Gly Ala Ala Cys 805 810 815 Gly Ala Cys Ala Thr Thr Thr Thr Thr Gly Ala Gly Gly Cys Cys Cys 820 825 830 Ala Gly Ala Ala Gly Ala Thr Ala Gly Ala Gly Thr Gly Gly Cys Ala 835 840 845 Cys Gly Ala Gly Thr Gly Ala 850 855 <210> 19 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 19 Met Met Arg Pro Ile Val Leu Val Leu Leu Phe Ala Thr Ser Ala Leu 1 5 10 15 Ala Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn 20 25 30 Pro Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile 35 40 45 Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu 50 55 60 Phe Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile 65 70 75 80 Ala Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr 85 90 95 Thr Pro Ile Thr Asn Val Pro Pro Glu Val Thr Val Leu Thr Asn Ser 100 105 110 Pro Val Glu Leu Arg Glu Pro Asn Val Leu Ile Cys Phe Ile Asp Lys 115 120 125 Phe Thr Pro Pro Val Val Asn Val Thr Trp Leu Arg Asn Gly Lys Pro 130 135 140 Val Thr Thr Gly Val Ser Glu Thr Val Phe Leu Pro Arg Glu Asp His 145 150 155 160 Leu Phe Arg Lys Phe His Tyr Leu Pro Phe Leu Pro Ser Thr Glu Asp 165 170 175 Val Tyr Asp Cys Arg Val Glu His Trp Gly Leu Asp Glu Pro Leu Leu 180 185 190 Lys His Trp Glu Phe Asp Ala Pro Ser Pro Leu Pro Glu Thr Thr Glu 195 200 205 Asn Gly Gly Gly Gly Ser Leu Glu Ile Arg Ala Ala Phe Leu Arg Gln 210 215 220 Arg Asn Thr Ala Leu Arg Thr Glu Val Ala Glu Leu Glu Gln Glu Val 225 230 235 240 Gln Arg Leu Glu Asn Glu Val Ser Gln Tyr Glu Thr Arg Tyr Gly Pro 245 250 255 Leu Gly Gly Gly Lys Gly Ser His His His His His His His His His 260 265 270 His <210> 20 <211> 822 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 20 Ala Thr Gly Ala Thr Gly Cys Gly Gly Cys Cys Cys Ala Thr Cys Gly 1 5 10 15 Thr Gly Cys Thr Gly Gly Thr Gly Cys Thr Gly Cys Thr Gly Thr Thr 20 25 30 Cys Gly Cys Cys Ala Cys Ala Thr Cys Thr Gly Cys Cys Cys Thr Gly 35 40 45 Gly Cys Cys Ala Thr Cys Ala Ala Ala Gly Ala Ala Gly Ala Ala Cys 50 55 60 Ala Thr Gly Thr Gly Ala Thr Cys Ala Thr Cys Cys Ala Gly Gly Cys 65 70 75 80 Cys Gly Ala Gly Thr Thr Cys Thr Ala Thr Cys Thr Gly Ala Ala Thr 85 90 95 Cys Cys Thr Gly Ala Cys Cys Ala Ala Thr Cys Ala Gly Gly Cys Gly 100 105 110 Ala Gly Thr Thr Thr Ala Thr Gly Thr Thr Thr Gly Ala Cys Thr Thr 115 120 125 Thr Gly Ala Thr Gly Gly Thr Gly Ala Thr Gly Ala Gly Ala Thr Thr 130 135 140 Thr Thr Cys Cys Ala Thr Gly Thr Gly Gly Ala Thr Ala Thr Gly Gly 145 150 155 160 Cys Ala Ala Ala Gly Ala Ala Gly Gly Ala Gly Ala Cys Gly Gly Thr 165 170 175 Cys Thr Gly Gly Cys Gly Gly Cys Thr Thr Gly Ala Ala Gly Ala Ala 180 185 190 Thr Thr Thr Gly Gly Ala Cys Gly Ala Thr Thr Thr Gly Cys Cys Ala 195 200 205 Gly Cys Thr Thr Thr Gly Ala Gly Gly Cys Thr Cys Ala Ala Gly Gly 210 215 220 Thr Gly Cys Ala Thr Thr Gly Gly Cys Cys Ala Ala Cys Ala Thr Ala 225 230 235 240 Gly Cys Thr Gly Thr Gly Gly Ala Cys Ala Ala Gly Cys Cys Ala 245 250 255 Ala Cys Cys Thr Gly Gly Ala Ala Ala Thr Cys Ala Thr Gly Ala Cys 260 265 270 Ala Ala Ala Gly Cys Gly Cys Thr Cys Cys Ala Ala Cys Thr Ala Thr 275 280 285 Ala Cys Thr Cys Cys Gly Ala Thr Cys Ala Cys Cys Ala Ala Thr Gly 290 295 300 Thr Ala Cys Cys Thr Cys Cys Ala Gly Ala Gly Gly Thr Ala Ala Cys 305 310 315 320 Thr Gly Thr Gly Cys Thr Cys Ala Cys Gly Ala Ala Cys Ala Gly Cys 325 330 335 Cys Cys Thr Gly Thr Gly Gly Ala Ala Cys Thr Gly Ala Gly Ala Gly 340 345 350 Ala Gly Cys Cys Cys Ala Ala Cys Gly Thr Cys Cys Thr Cys Ala Thr 355 360 365 Cys Thr Gly Thr Thr Thr Cys Ala Thr Cys Gly Ala Cys Ala Ala Gly 370 375 380 Thr Thr Cys Ala Cys Cys Cys Cys Ala Cys Cys Ala Gly Thr Gly Gly 385 390 395 400 Thr Cys Ala Ala Thr Gly Thr Cys Ala Cys Gly Thr Gly Gly Cys Thr 405 410 415 Thr Cys Gly Ala Ala Ala Thr Gly Gly Ala Ala Ala Ala Cys Cys Thr 420 425 430 Gly Thr Cys Ala Cys Cys Ala Cys Ala Gly Gly Ala Gly Thr Gly Thr 435 440 445 Cys Ala Gly Ala Gly Ala Cys Ala Gly Thr Cys Thr Thr Cys Cys Thr 450 455 460 Gly Cys Cys Cys Ala Gly Gly Gly Ala Ala Gly Ala Cys Cys Ala Cys 465 470 475 480 Cys Thr Thr Thr Thr Cys Cys Gly Cys Ala Ala Gly Thr Thr Cys Cys 485 490 495 Ala Cys Thr Ala Thr Cys Thr Cys Cys Cys Cys Thr Thr Cys Cys Thr 500 505 510 Gly Cys Cys Cys Thr Cys Ala Ala Cys Thr Gly Ala Gly Gly Ala Cys 515 520 525 Gly Thr Thr Thr Ala Cys Gly Ala Cys Thr Gly Cys Ala Gly Gly Gly 530 535 540 Thr Gly Gly Ala Gly Cys Ala Cys Thr Gly Gly Gly Gly Cys Thr Thr 545 550 555 560 Gly Gly Ala Thr Gly Ala Gly Cys Cys Thr Cys Thr Thr Cys Thr Cys 565 570 575 Ala Ala Gly Cys Ala Cys Thr Gly Gly Gly Ala Gly Thr Thr Thr Gly 580 585 590 Ala Thr Gly Cys Thr Cys Cys Ala Ala Gly Cys Cys Cys Thr Cys Thr 595 600 605 Cys Cys Cys Ala Gly Ala Gly Ala Cys Thr Ala Cys Ala Gly Ala Gly 610 615 620 Ala Ala Cys Gly Gly Cys Gly Gly Cys Gly Gly Ala Gly Gly Cys Ala 625 630 635 640 Gly Cys Cys Thr Gly Gly Ala Ala Ala Thr Cys Ala Gly Ala Gly Cys 645 650 655 Cys Gly Cys Cys Thr Thr Cys Cys Thr Gly Cys Gly Gly Cys Ala Gly 660 665 670 Ala Gly Ala Ala Ala Cys Ala Cys Cys Gly Cys Cys Cys Thr Gly Ala 675 680 685 Gly Ala Ala Cys Cys Gly Ala Ala Gly Thr Gly Gly Cys Cys Gly Ala 690 695 700 Gly Cys Thr Gly Gly Ala Ala Cys Ala Gly Gly Ala Ala Gly Thr Gly 705 710 715 720 Cys Ala Gly Cys Gly Gly Cys Thr Gly Gly Ala Ala Ala Ala Cys Gly 725 730 735 Ala Gly Gly Thr Gly Thr Cys Cys Cys Ala Gly Thr Ala Cys Gly Ala 740 745 750 Gly Ala Cys Ala Ala Gly Ala Thr Ala Cys Gly Gly Cys Cys Cys Thr 755 760 765 Cys Thr Gly Gly Gly Ala Gly Gly Cys Gly Gly Cys Ala Ala Gly Gly 770 775 780 Gly Cys Thr Cys Thr Cys Ala Cys Cys Ala Cys Cys Ala Cys Cys Ala 785 790 795 800 Thr Cys Ala Cys Cys Ala Thr Cys Ala Thr Cys Ala Thr Cys Ala Cys 805 810 815 Cys Ala Thr Thr Gly Ala 820 <210> 21 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 21 Cys Ala Ala Ser Arg Glu Ser Lys Trp Ser Ser Tyr Asn Ser Pro Leu 1 5 10 15 His Phe <210> 22 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 22 Cys Ala Asp Leu Ser Gly Gly Tyr Asn Lys Leu Ile Phe 1 5 10 <210> 23 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 23 Cys Ala Ser Ser Pro Thr Leu Gly Thr Asp Thr Gin Tyr Phe 1 5 10 <210> 24 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 24 Cys Ala Tyr Arg Ser Phe Leu Asn Ala Gly Gly Thr Ser Tyr Gly Lys 1 5 10 15 Leu Thr Phe

Claims

1. An HLA class II molecule comprising a DRβ chain, wherein the DRβ chain comprises an extracellular domain, wherein the amino acid sequence of the extracellular domain of the DRβ chain is shown in SEQ ID NO:

3.

2. An HLA class II molecule comprising a DRβ chain, wherein the DRβ chain comprises an extracellular domain, wherein the amino acid sequence of the extracellular domain of the DRβ chain is shown in SEQ ID NO:

12.

3. The HLA class II molecule of claim 1 or 2, wherein the beta chain of the HLA class II molecule comprises a DR2, DR3, DR4, DR5, DR6, DR7, DR8, DR9, DR10, DR11, DR12, DR13, DR14, DR15, or DR16 allele.

4. The HLA class II molecule of claim 1 or 2, wherein the β chain of the MHC class II molecule comprises an HLA allele selected from the group consisting of: DRB1*01, DRB1*03, DRB1*04, DRB1*07, DRB1*08, DRB1*09, DRB1*10, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*15, and DRB1*16. The HLA class II molecule according to claim 1 , further comprising an α chain. The HLA class II molecule of claim 5 , wherein the α chain of the MHC class II molecule comprises an HLA-DRA1*01 allele.

7. The HLA class II molecule of claim 1 or 2, wherein the DR β chain further comprises one or more leucine zipper sequences.

8. The HLA class II molecule according to claim 7, wherein the amino acid sequence of the DRβ chain is shown in SEQ ID NO:

4.

9. The HLA class II molecule of claim 1 or 2, wherein the beta chain of the HLA class II molecule comprises a DR1, DR3, DR4, DR7, DR8, DR9, DR10, DR11, DR12, DR13, DR14, DR15, or DR16 allele.

10. The HLA class II molecule of claim 1 or 2, wherein the β chain of the MHC class II molecule comprises an HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*06, HLA-DRB1*07, HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*10, HLA-DRB1*11, HLA-DRB1*12, HLA-DRB1*13, HLA-DRB1*14, HLA-DRB1*15, or HLA-DRB1*16 allele.

11. The HLA class II molecule of claim 5, wherein the α chain of the MHC class II molecule comprises an HLA-DRA1*01 allele.

12. The HLA class II molecule according to claim 5, wherein the amino acid sequence of the DRα chain is shown in SEQ ID NO: 6 or 8.

13. The HLA class II molecule of claim 1 or 2, wherein the DR β chain has increased affinity for CD4 protein compared to a reference HLA class II molecule, wherein the reference HLA class II molecule comprises a DR β chain comprising (i) a leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 and / or (ii) a valine at a position corresponding to amino acid residue 143 of SEQ ID NO:

1.

14. The HLA class II molecule of claim 13, wherein the increased affinity is at least 1.5-fold.

15. The HLA class II molecule of claim 1 or 2, wherein the DRβ chain is associated with a cell membrane. The HLA class II molecule of claim 1 , wherein the DR β chain is not associated with a cell membrane.

17. The HLA class II molecule of claim 1 or 2, wherein the DRβ chain does not comprise the transmembrane domain of the full-length DRβ chain.

18. The HLA class II molecule of claim 5, wherein the DR α chain is associated with a cell membrane. The HLA class II molecule of claim 5 , wherein the DR α chain is not associated with a cell membrane.

20. The HLA class II molecule of claim 5, wherein the DRα chain comprises the extracellular domain of a full-length DRα chain.

21. The HLA class II molecule of claim 20, wherein the DRα chain does not comprise the transmembrane domain of the full-length DRα chain.

22. The HLA class II molecule of claim 1 or 2, wherein the DR β chain is linked to or associated with an inert particle.

23. The HLA class II molecule of claim 22, wherein the inert particles are beads.

24. The HLA class II molecule of claim 22, wherein the inert particle is a nanoparticle.

25. The HLA class II molecule of claim 24, wherein the nanoparticles are selected from PEGylated iron oxide, chitosan, dextran, gelatin, alginate, liposomes, starch, branched polymers, carbon-based carriers, polylactic acid, poly(cyano)acrylate, polyethyleneimine, block copolymers, polycaprolactone, SPIONS, USPIONS, Cd / Zn-selenide, or silica nanoparticles.

26. The HLA class II molecule of claim 22, wherein the nanoparticles are PEGylated iron oxide nanoparticles.

27. The HLA class II molecule of claim 1 or 2, wherein the DR β chain comprises a signal peptide.

28. The HLA class II molecule of claim 5, wherein the DR α chain further comprises a signal peptide.

29. The HLA class II molecule of claim 27 or 28, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO:

9.

30. A nucleic acid molecule encoding the DR β chain of any one of claims 1 to 29. The nucleic acid molecule of claim 30 , further encoding the DR α chain of claim 5 .

32. A vector comprising the nucleic acid molecule of claim 30 or 31.

33. A cell comprising the HLA class II molecule of any one of claims 1 to 29, the nucleic acid molecule of claim 30 or 31, or the vector of claim 32.

34. The cell of claim 33, which is a mammalian cell or an insect cell.

35. The cell of claim 33 or 34, which is selected from K562 cells, T2, HEK293, A375, SK-MEL-28, Me275, COS, fibroblasts, or any combination thereof.

36. The cell of claim 33 or 34, which is a HEK293T cell.

37. The cell of claim 33 or 34, which is a tumor cell.

38. The cell of claim 33 or 34, which lacks endogenous MHC class II DR β chain expression.

39. The cell of claim 33 or 34, which lacks endogenous MHC class II DR alpha chain expression.

40. A method for identifying a T cell receptor capable of binding an epitope in an MHC class II complex, the method comprising pulsing the cells of any one of claims 33 to 39 with one or more peptides comprising the epitope, and stimulating one or more CD4 T cells with an APC. + T cells.

41. The HLA class II molecule of claim 1 or 2, which has a K of less than about 20 μM. D Binds to CD4.

42. The HLA class II molecule of claim 1 or 2, which has a K of about 14 μM or less. D Binds to CD4.

43. A complex comprising an HLA class II molecule according to any one of claims 1 to 29, 41 and 42 and a peptide, wherein the peptide is selected from the group consisting of: NY-ESO1 91-110 、Bet V 1 142-153 、HIV Gag 293-312 , HA 306-318 、Flu-HA 5-24 、Flu-HA 117-136 、Flu-HA 232-251 、Flu-HA 268-287 、Flu-HA 306-318 、HSD17B12 225-244 LY6K 99-118 and any combination thereof.

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