Peptide-HLA complexes and methods of producing the same

Abstract

To provide a method of producing an HLA class I molecule complexed to a peptide.SOLUTION: For example, there is provided a method of producing an HLA class I molecule complexed to a pre-selected peptide, the method comprising: (a) providing a mammalian derived HLA class I molecule complexed to an existing peptide; (b) incubating, in vitro, the HLA class I molecule complexed to the existing peptide with the pre-selected peptide, where the pre-selected peptide is at a concentration sufficient to replace the existing peptide to produce the HLA class I molecule complexed to the pre-selected peptide; and the HLA class I molecule comprises α1, α2, α3 and β2m domains.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to the field of human leukocyte antigen (HLA) class I molecules, and more particularly to methods for producing HLA class I molecules conjugated to peptides. [Background technology]

[0002] Analysis of antigen-specific T cells using flow cytometry with peptide-MHC (pMHC) multimers has been established as a standard technique in immunology (1, 2). These reagents allow tracking and phenotypic analysis of antigen-specific T cells during immune responses associated with infection, autoimmunity, GVHD, and cancer.

[0003] The αβ T cell antigen receptor (TCR) in T cells recognizes peptide antigens presented by MHC class I or II molecules on the cell surface (Non-Patent Documents 3, 4). The interaction between TCR and pMHC is very weak, and monomeric soluble pMHC generally cannot stably associate with the cell surface of T cells bearing the cognate TCR. pMHC multimers, in the form of avidin-biotin-based pMHC tetramers, were first introduced by Mark Davis et al. in 1996 and immediately transformed the analysis of antigen-specific T cells (Non-Patent Document 5). pMHC multimers have been used in many studies, and some commercial vendors, such as BD BioSciences™, ProImmune™, Immudex™, and TC matrix™, sell various forms of pMHC multimers. pMHC multimers can be used in conjunction with combinations of antibodies specific for other cell surface molecules (Non-Patent Document 6). Thus, simultaneous staining for TCR and immunoaccessory molecules allows for the classification of antigen-specific T cells into various phenotypically distinct subsets, which can be used to characterize antigen-specific T cells with respect to antigen exposure, effector function, and status. Summary of the Invention [Problem to be solved by the invention]

[0004] Expression in E. coli is the preferred method for producing MHC class I proteins and can provide large quantities of highly purified protein (http: / / tetramer.yerkes.emory.edu / support / protocols). Unlike class II molecules, most class I molecules are unstable due to the lack of peptide in the groove (Non-Patent Document 7). Therefore, in virtually all cases, a synthetic peptide of interest is attached to the MHC class I molecule, and the class I expression process is coupled to the peptide binding process to produce the complete pMHC complex. Bacterial systems have several known problems. For some HLA class I genes, such as HLA-B alleles, bacterial pHLA production is difficult, in part, due to poor refolding (Non-Patent Documents 8, 9). Although glycosylation on class I proteins is not required for the interaction between pMHC and the cognate TCR, the lack of sugar moieties on bacterially expressed MHC class I proteins can have a negative impact on their stability. Furthermore, bacterially expressed pMHC proteins refolded in vitro may not have the exact same conformation as those produced in mammals and refolded in vivo. While peptide exchange of the generated intact pMHC proteins is possible in vitro, it requires multiple complex steps (Non-Patent Documents 10-12). Therefore, high-throughput production of pMHC proteins is labor-intensive, cumbersome, and not widely available. Finally, the pMHC-TCR affinity required for pMHC multimer binding has been shown to exceed that required for T cell activation (Non-Patent Document 13). The observed difference in affinity thresholds means that current pMHC tetramer staining cannot detect all antigen-specific T cells, especially those with low affinity. When pMHC multimers are used to stain autoantigen-specific T cells associated with immune responses in autoimmunity and cancer, the inability to stain all cognate T cells expressing TCRs with a wide range of affinities, which tend to express low-affinity TCRs, is likely to be a serious problem. [Means for solving the problem]

[0005] According to one embodiment, a method for producing an HLA class I molecule conjugated to a preselected peptide is provided by first providing an HLA class I molecule from a mammal conjugated to a pre-existing peptide. The HLA class I molecule conjugated to the pre-existing peptide is then incubated in vitro with the pre-selected peptide at a concentration sufficient to replace the pre-existing peptide, thereby producing an HLA class I molecule conjugated to the pre-selected peptide. The HLA class I molecule contains α1, α2, α3, and β2m domains.

[0006] According to a further aspect, a kit for producing an HLA class I molecule conjugated to a preselected peptide is provided, the kit comprising an HLA class I molecule from a mammal conjugated to a pre-existing peptide and instructions corresponding to the above method. In some embodiments, the kit further comprises a preselected peptide.

[0007] According to a further aspect, there is provided a polypeptide comprising the α1, α2, α3 domains of an HLA class I molecule, a signal peptide at the N-terminus of the HLA class I molecule, and a 6xHis tag joined by a GS linker at the C-terminus.

[0008] According to a further aspect, there is provided a nucleic acid encoding the above polypeptide.

[0009] According to a further aspect, there is provided a vector comprising the above nucleic acid.

[0010] According to a further aspect, there is provided a mammalian cell transfected with the above vector.

[0011] According to a further aspect, there is provided a compound comprising the above polypeptide conjugated to a β2m domain.

[0012] According to a further aspect, there is provided a multimer of at least two of the above compounds.

[0013] In one embodiment, a method of screening / selecting a population of T cells for antigen-specific T cells that recognize a preselected peptide antigen comprises providing an HLA class I molecule from a mammal conjugated to a preselected peptide, and screening the population of T cells for antigen-specific T cells that bind to the HLA class I molecule from the mammal conjugated to the preselected peptide. [Brief explanation of the drawings]

[0014] Embodiments of the present invention may be best understood by referring to the following description and accompanying drawings, in which: [Figure 1] FIG. 1 is a schematic diagram showing the general structure of an HLA class I molecule conjugated to a peptide. [Figure 2] FIG. 1 shows the production of monomeric pHLA displaying a peptide of interest by in vitro peptide exchange. [Figure 3] FIG. 1 shows a timeline chart of harvesting, peptide binding, and dimerization of pHLA for staining antigen-specific T cells. [Figure 4] FIG. 1 shows staining data showing peptide exchange occurring in supernatant samples. [Figure 5] FIG. 1 shows staining data showing A2 / MART1 monomer staining in high avidity A2 / MART1 T cells but not in A2 / NY-ESO-1 T cells. [Figure 6] FIG. 1 shows staining data showing A2 / NY-ESO-1 monomer staining in high avidity A2 / NY-ESO-1 T cells but not in A2 / MART1 T cells. [Figure 7] FIG. 1 shows staining data showing A2 / MART1 dimer staining in A2 / MART1 T cells but not in A2 / NY-ESO-1 T cells. [Figure 8]FIG. 1 shows staining data showing A2 / NY-ESO-1 dimer staining in A2 / NY-ESO-1 T cells but not in A2 / MART1 T cells. [Figure 9] FIG. 1 shows staining data showing that an embodiment of the dimer stains the lower affinity A2 / MART1 TCR better than the known pentamer. [Figure 10] FIG. 1 shows staining data showing that the A24Q115E-Kb dimer stains the low affinity A24 / WT1 TCR better than the prior art A24 / WT1 tetramer. [Figure 11] FIG. 1 shows staining data showing B35Q115E-Kb dimer staining of B*35:01 / EBNA-1407-417 TCR (clone TK3). [Figure 12] FIG. 10 shows staining data showing high-throughput A2 dimer staining of TILs. [Figure 13] FIG. 1 shows B*44:05 dimer staining for B*44:05 / EBNA-6281-290 TCR. [Figure 14] Figure 1 shows C*07:02 / MAGE-A1289-297 and C*07:02 / MAGE-A12170-178 dimers stained for their respective TCRs. [Figure 15] FIG. 1 shows A2+ melanoma TILs. [Figure 16] High-throughput A2 dimer staining of TILs (TIL:M25 TIL16 REP1 2E7 2016-9-15). [Figure 17] High-throughput A2 dimer staining of TILs (TIL:M31 TIL3 REP1A 2E7 2015-06-03). [Figure 18] High-throughput A2 dimer staining of TILs (TIL:M37 TIL3 REP1B 2E7 2015-06-03). [Figure 19]High-throughput A2 dimer staining of TILs (TIL:M40 TIL3 REP1A 2E7 2015-06-04). [Figure 20] FIG. 1 shows high-throughput A2 dimer staining of TILs (TIL:M66 YT REP1A D14 2E7 2012-02-01). [Figure 21] High-throughput A2 dimer staining of TILs (TIL:M96 YT REP1A 2E7 2015-06-04). [Figure 22] Figure 1 shows IFN-γ ELISPOT assay (TIL:M25 TIL16 REP1 2E7 2016-9-15). [Figure 23] Figure 1 shows IFN-γ ELISPOT assay (TIL:M31 TIL3 REP1A 2E7 2015-06-03). [Figure 24] FIG. 1 shows IFN-γ ELISPOT assay (TIL:M37 TIL3 REP1B 2E7 2015-06-03). [Figure 25] Figure 1 shows IFN-γ ELISPOT assay (TIL:M40 TIL3 REP1A 2E7 2015-06-04). [Figure 26] Figure 1 shows IFN-γ ELISPOT assay (TIL:M66 YT REP1A D14 2E7 2012-02-01). [Figure 27] Figure 1 shows IFN-γ ELISPOT assay (TIL:M96 YT REP1A 2E7 2015-06-04). [Figure 28] FIG. 1 shows an overview of A2 dimer staining. [Figure 29] FIG. 1 shows the enrichment of dimer-positive TILs. DETAILED DESCRIPTION OF THE INVENTION

[0015] We have developed a novel technology that enables high-throughput production of mammalian-derived peptide / HLA class I (pHLA) multimers capable of staining low-affinity TCRs. One application of this technology is to generate personalized pHLA reagents that enable high-throughput measurement of anti-tumor T cell responses in cancer patients.

[0016] According to one aspect, a method for producing an HLA class I molecule conjugated to a preselected peptide is provided by first providing an HLA class I molecule from a mammal conjugated to a pre-existing peptide. The HLA class I molecule conjugated to the pre-existing peptide is then incubated in vitro with the pre-selected peptide at a concentration sufficient to replace the pre-existing peptide, thereby producing an HLA class I molecule conjugated to the pre-selected peptide. The HLA class I molecule comprises α1, α2, α3, and β2m domains. In some embodiments, the HLA class I molecule is soluble.

[0017] human leukocyte antigen The HLA system is a complex of genes that encode human major histocompatibility complex (MHC) proteins. These cell surface proteins are involved in regulating the immune system in humans. HLA genes are highly polymorphic, and different classes have distinct functions. HLA class I genes, which encode MHC class I molecules, function to display or present peptide fragments of non-self or self proteins from within cells to cytotoxic T cells.

[0018] As used herein, the phrase "HLA class I molecule" refers to a protein molecule derived from expression of a wild-type or mutant HLA class I gene encoding an MHC class I molecule. A schematic diagram of the general structure of an HLA class I molecule, with its α1, α2, α3, and β2m domains, is shown in Figure 1.

[0019] The schematic also illustrates a peptide complexed to an HLA class I molecule. As used herein, the term "peptide" refers to a peptide fragment capable of forming a complex with an HLA class I molecule and being displayed or presented by the HLA class I molecule. Such peptides are well described in the art. Generally, these particular peptides are about 8-15 amino acids in length, but may vary in length between 8-10, 7-11, or 6-12 amino acids.

[0020] For some HLA class I genes, bacterial production of pHLA is difficult, in part due to insufficient refolding. Furthermore, bacterially expressed pMHC proteins refolded in vitro may not have the exact same conformation as those produced in mammals and refolded in vivo. As used herein, the term "mammalian-derived" refers to the production of molecules using mammalian cell systems well known in the art, such as human cell lines (e.g., Hela, HEK293, HEK293T, and their derivatives), monkey cell lines (e.g., CV-1, COS, and their derivatives), mouse cell lines (e.g., NIH3T3 and its derivatives, NS-1, and its derivatives), and hamster cell lines (e.g., BHK, CHO, and their derivatives). In one embodiment, a human cell line is used. In one example, the HEK293T cell line can be used. The HLA class I molecule conjugated to a pre-existing peptide is produced by mammalian cells transfected with a soluble HLA class I molecule, in which the β2m domain can be endogenous or exogenous. In a preferred embodiment, the β2m domain is exogenous and encoded on a second vector.

[0021] In some embodiments, the soluble HLA class I molecule comprises a signal peptide that directs secretion of the HLA class I molecule to the outside of the mammalian cell. In other embodiments, the soluble HLA class I molecule conjugated to a pre-existing peptide is provided in the culture supernatant of the mammalian cell.

[0022] HLA class I genes HLA class I genes are a family of genes. HLA class I molecules can be HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.

[0023] As used herein, "HLA-A" refers to a protein molecule derived from expression of the HLA-A gene. "HLA-B" refers to a protein molecule derived from expression of the HLA-B gene. "HLA-C" refers to a protein molecule derived from expression of the HLA-C gene. "HLA-D" refers to a protein molecule derived from expression of the HLA-D gene. "HLA-E" refers to a protein molecule derived from expression of the HLA-E gene. "HLA-F" refers to a protein molecule derived from expression of the HLA-F gene. "HLA-G" refers to a protein molecule derived from expression of the HLA-G gene. All genes HLA-A through HLA-G are part of the HLA class I family of genes.

[0024] Amino acid sequence of HLA class I molecules HLA class I molecules can have many amino acid sequence variants.

[0025] In some embodiments, the α3 domain of the HLA class I molecule is a mouse Kbα3 domain (designated Kb). In other embodiments, in the α2 domain of the HLA class I molecule, Gln is substituted with Glu at position 115 (designated Q115E).

[0026] Exemplary HLA class I molecules include, but are not limited to, the following:

[0027] The HLA class I molecule may be HLA-A and comprise the α1, α2, and α3 domains of either SEQ ID NO: 6 or 12. In other embodiments, the HLA-A α1, α2, and α3 domains may be wild-type, such as SEQ ID NO: 2 or 14, respectively. In yet other embodiments, the HLA class I molecule has wild-type α1 and α2 domains and a mouse Kbα3 domain, as in SEQ ID NO: 4 or 10, respectively. Any combination of the above is also possible.

[0028] The HLA class I molecule may be HLA-B and comprise the α1, α2, and α3 domains of any of SEQ ID NOs: 14, 16, 18, 20, or 22. As with the exemplary HLA-A molecule, the α1, α2, and α3 domains may be wild-type or may be selected variants such as Kb and Q115E, or any combination thereof.

[0029] The HLA class I molecule may be HLA-C and comprise the α1, α2, and α3 domains of any of SEQ ID NOs: 24, 26, 28, or 30. As with the exemplary HLA-A molecule, the α1, α2, and α3 domains may be wild-type or may be selected variants such as Kb and Q115E, or any combination thereof.

[0030] In still other embodiments, the HLA class I molecule comprises α1, α2, and α3 domains with a β2m domain as described herein.

[0031] Multimers The HLA class I molecule may be multimerized. According to a further embodiment, the method further comprises multimerizing the HLA class I molecule, preferably into one of a dimer, a trimer, a tetramer, and a pentamer.

[0032] In some embodiments, HLA class I molecules are dimerized using antibodies that recognize corresponding tags on HLA class I molecules.In further embodiments, the tag is preferably a 6xHis tag at the C'-end of α3 domain, which is linked by a flexible linker, more preferably a GS linker.Other suitable tags for antibody binding are known in the art.There are many examples of acceptable tags, including AviTag, calmodulin tag, polyglutamate tag, His tag, Myc tag, and VSV tag.There are many examples of acceptable flexible linkers, including Chen et al., Adv Drug Deliv Rev. 2013 Oct 15;65(10):1357-1369.

[0033] Kits and Reagents According to a further aspect, a kit for producing an HLA class I molecule conjugated to a preselected peptide is provided, the kit comprising an HLA class I molecule from a mammal conjugated to a pre-existing peptide and instructions corresponding to the above method. In some embodiments, the kit further comprises a preselected peptide.

[0034] According to a further aspect, there is provided a polypeptide comprising the α1, α2, and α3 domains of an HLA class I molecule, a signal peptide at the N-terminus of the HLA class I molecule, and a 6xHis tag joined to the C-terminus by a GS linker. In some embodiments, the polypeptide is any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.

[0035] According to a further aspect, there is provided a nucleic acid encoding the above polypeptide.

[0036] According to a further aspect, there is provided a vector comprising the above nucleic acid.

[0037] According to a further aspect, there is provided a mammalian cell transfected with the above vector. In some embodiments, the mammalian cell further comprises a second vector encoding β2m.

[0038] According to a further aspect, there is provided a compound comprising the above polypeptide complexed with a β2m domain.

[0039] According to a further aspect, there is provided a multimer of at least two of the above compounds. In some embodiments, the at least two compounds are dimerized by an antibody that recognizes the 6xHis tag.

[0040] The following examples are illustrative of various aspects of the present invention and are not intended to limit the broad aspects of the invention disclosed herein.

[0041] Screening and selection of T cells, including tumor-infiltrating lymphocytes It is known that T cells can be screened / selected using HLA class I molecules conjugated to preselected peptides. Peptide antigens are recognized via this T cell receptor. Advantageously, the mammalian-derived HLA class I molecules described herein allow those skilled in the art to swap out existing (or holder) peptides with preselected peptides of interest. This was not possible with existing bacterial-derived HLA class I molecules. Rather, existing bacterial-derived HLA class I molecules had to be produced, denatured, and then refolded with the peptide antigen of interest.

[0042] Therefore, the mammal-derived HLA class I molecules of the present invention represent a streamlined and more flexible procedure for easily producing molecules capable of presenting peptide antigens. For example, the mammal-derived HLA class I molecules of the present invention can be produced in advance and replaced with a holder peptide before use. Furthermore, the mammal-derived HLA class I molecules of the present invention do not require refolding and are glycosylated, so they are likely to be representative of natural HLA class I molecules.

[0043] Thus, in one aspect, there is provided a method of screening / selecting a population of T cells for antigen-specific T cells that recognize a preselected peptide antigen, the method comprising providing an HLA class I molecule from a mammal conjugated to a preselected peptide, and screening the population of T cells for antigen-specific T cells that bind to the HLA class I molecule from a mammal conjugated to the preselected peptide.

[0044] In some embodiments, the method further comprises first providing an HLA class I molecule from a mammal conjugated to a holder peptide, and incubating in vitro the HLA class I molecule conjugated to the holder peptide with a preselected peptide, wherein the preselected peptide is at a concentration sufficient to replace the existing peptide to produce an HLA class I molecule conjugated to the preselected peptide.

[0045] In some embodiments, the mammalian-derived HLA class I molecule complexed with the preselected peptide is prepared using the methods described herein.

[0046] In some embodiments, the screening comprises flow cytometry.

[0047] In some embodiments, the HLA class I molecule is complexed with a holder peptide and comprises any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30, and preferably has a β2m domain.

[0048] In some embodiments, the HLA class I molecule complexed to the holder peptide comprises a polypeptide described herein, preferably having a β2m domain.

[0049] In some embodiments, the methods can be used to screen / select T cell populations associated with cancers including adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain / cns tumors, breast cancer, Castleman's disease, cervical cancer, colon / rectal cancer, uterine cancer, esophageal cancer, family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (gist), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, renal cancer, pharyngeal and hypopharyngeal cancer, leukemia (acute lymphocytic, acute myeloid, chronic lymphocytic, chronic myelogenous, chronic myelomonocytic), liver cancer, lung cancer (non-small cell, small cell, lung carcinoid tumors), lymphoma, lymphoma of the skin, malignant mesothelial tumors, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma - adult soft tissue cancer, skin cancer (basal and squamous cell, melanoma, Merkel cell), small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, or Wilms' tumor.

[0050] In some embodiments, the antigen-specific T cells that recognize the preselected peptide antigen are tumor-infiltrating lymphocytes. In some embodiments, the preselected peptide antigen is associated with cancer.

[0051] Example material and method peptide Synthetic peptides were purchased from ProImmune, Genway Biotech, and GenScript. The peptides used were the A2-restricted heteroclitic MART1. 26-35 (ELAGIGILTV), Heterocritic NY-ESO-1 157-165 (SLLMWITQV), A24-restricted heteroclitic WT1 235-243 (CYTWNQMNL), B35-restricted wild-type EBNA-1 407-417 (HPVGEADYFEY) peptide, B44-restricted wild-type EBNA-6 281-290 (EENLLDFVRF), C7-restricted wild-type MAGE-A1 289-297 (RVRFFFPSL), and C7-restricted wild-type MAGE-A12 170-178 (VRIGHLYIL) peptide. The A2 peptides used to stain TILs are listed in Table 1 below.

[0052] Table 1 [Table 1] [Table 2] [Table 3]

[0053] Cells and cDNA HEK293T cells were obtained from the American Type Culture Collection. HLA-A2 harboring metastatic melanoma +TILs isolated from patients were expanded in vitro as previously reported (Non-Patent Document 14). Appropriate informed consent and institutional review board approval were obtained. All clonotype TCR genes were reconstituted in Jurkat 76 / CD8 cells or primary T cells as previously described. cDNAs were fused to a puromycin resistance gene via an internal ribosome entry site (Non-Patent Documents 15, 16). Transduced cells were isolated by puromycin selection. All cDNAs were cloned into pMX vectors and transduced using a 293GPG cell-based retroviral system (Non-Patent Documents 16-19).

[0054] Flow cytometry analysis The following surface antigens were recognized by mAbs: β2m (551337, BD BioSciences) and His (ab72467, Abcam). Mouse isotype controls were obtained from BD BioSciences. Surface molecule staining was performed as described elsewhere (Non-Patent Documents 16, 20).

[0055] Immunoblotting For immunoblotting, cells were extracted with ice-cold Nonidet P-40 (NP-40) extraction buffer (20 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 150 mM NaCl, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1% NP-40, 1 mM PMSF, and 1 μg / ml aprotinin). Cell extracts were centrifuged at 10,000 g for 10 minutes at 4°C, separated by Tris-glycine SDS-PAGE, and subsequently electrophoretically transferred to an Immobilon-P membrane (Millipore). After blocking with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, the membrane was incubated overnight at 4°C with the indicated mouse anti-His mAb (sc-53073, Santa Cruz Biotechnology), washed, and incubated with an HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Promega) for 1 hour at room temperature. Signals were detected by enhanced chemiluminescence (GE Healthcare).

[0056] Results and Discussion Structure of a soluble monomeric peptide / HLA class I (pHLA) complex HLA class I molecules are heterodimers consisting of two noncovalently linked polypeptide chains, α and β2-microglobulin (β2m). The α chain is highly polymorphic, while the β2m subunit is monotypic. The HLA class I α1 and α2 domains form a peptide groove of 8–10 amino acids in length. The α3 domain, which contains the transmembrane domain, binds to β2m. TCRs on the surface of cytotoxic T cells recognize peptides presented by the HLA class I α2 and α2 domains to interrogate antigenicity, and the CD8 coreceptor binds to the α2 and β3 domains to stabilize the interaction between the TCR and pHLA. Therefore, enhancing the interaction between CD8 and HLA class I improves the strength of the interaction between pHLA and the cognate TCR.

[0057] Mouse K in the HLA class I α3 domain bSubstitution by the α3 domain (hereafter referred to as class IK b The substitution of a Gln(Q) residue at position 115 of the α2 domain with a Glu(E) residue (hereafter referred to as class I) has been shown to increase the interaction between class I and CD8 by 10-fold. Q115E The interaction is further improved by 1.5-fold by using a soluble HLA class I fusion protein (referred to as "K") (Non-Patent Documents 21, 22). Soluble class I-wt was generated by fusing the extracellular domain of wild-type (wt) HLA class I with a Gly-Ser (GS) flexible linker followed by a 6xHis tag. Soluble class IK b and class IQ115E -K b The soluble class I-wt and class IK proteins used in this study were also produced in the same manner. b and Class I Q115E -K b The nucleotide and amino acid sequences of the gene are listed below.

[0058] Production of soluble monomeric pHLA complexes using mammalian cells HEK293T cells were first transfected using pMX vectors and a 293GPG cell-based retroviral system with the β2m gene, followed by the soluble HLA class IK b or HLA class I Q115E -K b The gene was transfected (Non-Patent Documents 16-19).

[0059] Enhancement of β2m expression by gene transfer Flow cytometry analysis after staining with β2m-specific mAb revealed that soluble forms of HLA-A2-K b or A2 Q115E -K b Enhanced expression of β2m was demonstrated in HEK293T cells stably transfected with the β2m gene. The HLA-A*02:01 (A2) gene, one of the most frequent HLA class I alleles, was used as a representative HLA class I gene. Using the same strategy, we generated HEK293T-derived cell lines stably expressing soluble forms of other class I genes.

[0060] Cellular expression of soluble monomeric peptide / HLA (pHLA) in HEK293T transfectants Soluble HLA-A2-Kb or A2 conjugated with or without the β2m gene Q115E Whole cell lysates from HEK293T cells stably expressing the HLA-A2-Kb gene were blotted with anti-His mAb as described above (Non-Patent Documents 23-25). b and A2 Q115E -K b The cellular expression of was demonstrated at the protein level.

[0061] Secretion of soluble monomeric pHLA complexes into the supernatant Soluble HLA-A2-K b or A2 Q115E -K b Supernatants from transfected HEK293T cells were harvested with or without the β2m gene and blotted with His-specific mAb. Bacterially expressed 6xHis-tagged HLA-A2 / heteroclitic MART1. 26-35 The indicated amount of monomer (NIH Tetramer Core Facility) was bound as a control. 10 μl of each supernatant was loaded per lane without concentration. Monomeric HLA-A2-K b and A2 Q115E -K b Secretion of the enzyme into the medium was confirmed. Monomeric pHLA complexes were secreted only when β2m was overexpressed.

[0062] Soluble HLA-A2-K without the β2m gene expressed in HEK293T cells b or A2 Q115E -K b When only the gene was transduced, soluble A2-K b and A2 Q115E -K b Secretion of β2m into the culture medium was not detected, indicating that endogenous β2m expression levels were significantly higher than those of ectopically expressed soluble A2-K. b and A2 Q115E -K bThis suggests that the amount of ATP produced was insufficient to allow secretion of ATP.

[0063] Production of monomeric pHLA conjugated with peptides of interest by in vitro peptide exchange Soluble HLA-A2-K produced by HEK293T transformants b and A2 Q115E -K b The containing supernatant was simply mixed at room temperature with the indicated concentrations of the A2-restricted peptide of interest for in vitro peptide exchange (see Figure 2 ).

[0064] Dimerization of monomeric pHLA complexes Soluble HLA class I in HEK293T conditioned medium Q115E -K b The monomers were dimerized using an anti-His mAb conjugated with a fluorescent dye such as phycoerythrin (PE) at a molar ratio of 2:1. Note that the soluble protein was fused with a 6xHis tag at the C-terminus.

[0065] Complete protocol for the production of dimeric pHLA complexes for staining antigen-specific T cells Soluble Monomer Class I Q115E -K b Stable HEK293T cell lines ectopically expressing β2m and β2m were established as described above. The stable cell lines were grown to confluence, and the medium was replaced. After 48 hours, the conditioned medium was harvested and used immediately or frozen until further use. For in vitro peptide exchange, the class I-restricted peptide of interest was bound to the supernatant at 37°C for 24 hours. The peptide-bound soluble monomeric class I peptide was then purified. Q115E -K b was dimerized with a fluorescent dye-conjugated anti-His mAb at 4° C. for 24 hours (see FIG. 3). Peptide exchange occurs in the supernatant by simple mixing.

[0066] Simply mix to dissolve A2 Q115E -K b A2 / MART1 to the monomer26-35 (ELAGIGILTV) or A2 / NY-ESO-1 157-165 The (SLLMWITQV) peptide was conjugated and dimerized with a PE-conjugated anti-His mAb, and used to stain human Jurkat76 / CD8 T cells expressing the clonotype-cognate TCR (see Figure 4). Jurkat76 / CD8 cells, which lack endogenous TCR expression, stably express the CD8α / β genes (Non-Patent Documents 26, 27). Soluble monomer A2 Q115E -K b stains high avidity antigen-specific T cells.

[0067] Soluble A2 by simple mixing Q115E -K b A2 / MART1 to the monomer 26-35 or A2 / NY-ESO-1 157-165 The peptide was conjugated and used directly, without dimerization, to stain Jurkat76 / CD8 T cells expressing the clonotype cognate TCR. Jurkat76 / CD8 cells expressing a high-avidity, but low-affinity TCR were stained with monomeric soluble A2 ligated with the cognate peptide. Q115E -K b The cells were stained with HCl (see Figures 5 and 6) (Non-Patent Document 27). Soluble dimer A2 Q115E -K b stains high and low avidity antigen-specific T cells.

[0068] Soluble monomer A2 Q115E -K b The supernatant containing A2 / MART1 was mixed by simple mixing. 26-35 or A2 / NY-ESO-1 157-165 The peptides were conjugated, dimerized with PE-conjugated anti-His mAb, and utilized to stain Jurkat 76 / CD8 T cells expressing the clonotype-cognate TCR (see Figures 7 and 8). Both the high-affinity and low-affinity TCRs expressed on Jurkat 76 / CD8 cells were stained with soluble dimeric A2 peptides. Q115E -K bThe staining was carried out by (Non-Patent Document 27). Soluble dimeric class I Q115E-Kb stains low affinity TCRs better than pentamers (ProImmune) or tetramers (NIH).

[0069] PE-conjugated soluble dimeric A2 Q115E -K b and A24 Q115E -K b A2 / MART1 respectively 26-35 and A24 / WT1 235-243 The (CYTWNQMNL) peptide was conjugated to the dimer. The conjugated dimer was used to stain Jurkat 76 / CD8 T cells expressing clonotype-cognate TCRs with various affinities (Non-Patent Documents 26, 27). Our dimer stained low-affinity TCRs better than the pentamer (ProImmune) and the NIH tetramer (see Figures 9 and 10). The pentamer was used according to the protocol provided by the vendor (https: / / www.proimmune.com / ecommerce / page.php?page=protocols). Tetramer staining was performed according to standard protocols published elsewhere (Non-Patent Documents 26, 27). Soluble dimeric HLA-B Q115E -K b also works in the same way.

[0070] Soluble monomeric HLA-B35 Q115E -K b , B35 / EBNA-1 407-417 (HPVGEADYFEY) peptide was conjugated and dimerized with a PE-conjugated anti-His mAb to stain Jurkat 76 / CD8 T cells expressing the clonotype cognate TCR (see Figure 11).

[0071] Soluble monomeric HLA-B44 Q115E -K b is B44 / EBNA-6 281-290(EENLLDFVRF) was bound and dimerized with PE-conjugated anti-His mAb and used to stain Jurkat 76 / CD8 T cells expressing the clonotype-cognate TCR (Fig. 13 ). Soluble dimeric HLA-C Q115E -K b also works in the same way.

[0072] Soluble monomeric HLA-C7 Q115E -K b is C7 / MAGE-A1 289-297 (RVRFFFPSL) peptide and C7 / MAGE-A12 170-178 The (VRIGHLYIL) peptide was conjugated and dimerized with a PE-conjugated anti-His mAb and used to stain Jurkat 76 / CD8 T cells expressing the clonotype-cognate TCR (Fig. 14). Staining of in vitro expanded tumor-infiltrating lymphocytes with a panel of soluble A2 dimers.

[0073] Peripheral T cells do not always reflect the immune response to tumors in cancer patients, and peripheral anti-tumor cell-mediated immunity often does not correlate with prognosis. In contrast, tumor-infiltrating lymphocytes (TILs) interact closely with tumor cells and are likely to reflect tumor-host interactions with a high degree of accuracy. The use of TILs as a graft for adoptive cell transfer therapy to treat cancer has been pioneered by the Rosenberg group at the National Cancer Institute (NCI) (28).

[0074] TILs are considered to be a polyclonal population of T cells with various antigen specificities (Non-Patent Document 29). To investigate the tumor specificity of TILs using our soluble dimer pHLA technique, we analyzed HLA-A2 T cells from nine patients with metastatic melanoma. +TILs were isolated from patients and expanded in vitro as previously reported (Non-Patent Document 14). A large panel of 8- to 11-mer peptides derived from proteins highly expressed by autologous tumor cells was predicted using publicly available algorithms previously reported (see Table 1) (Non-Patent Documents 18, 23, 30). The predicted A2 peptides were bound to soluble dimeric A2 peptides. Q115E -K b The library was produced as described above and used to stain TILs (see Figures 12 and 15-21). 476-484 and A2 / MART1 26-35 Pentamer (ProImmune) was used as a negative and positive control, respectively. The results showed that in vitro expanded TILs were reactive to MART1, one of the established melanoma-associated antigens (http: / / www.uniprot.org / uniprot / Q16655).

[0075] dimer + T cell functional assays Using ELISPOT assays, all six TIL samples that stained positive for dimers were confirmed to secrete A2-restricted peptide-specific IFN-γ. PVDF plates (Millipore, Bedford, MA) were coated with a capture mAb (1D1K; MABTECH, Mariemont, OH). TILs were cultured at 2 × 10 in the presence of each peptide. 4 The wells were incubated with T2 cells for 20–24 hours at 37°C. The plates were washed and incubated with a biotin-conjugated detection mAb (7-B6-1; MABTECH). Then, HRP-conjugated SA (Jackson ImmunoResearch) was added to generate IFN-γ spots. The reaction was stopped by thorough rinsing with cold tap water. ELISPOT plates were scanned and counted using an ImmunoSpot plate reader and ImmunoSpot version 5.0 software (Cellular Technology Limited, Shaker Heights, OH) (Figures 22–27).

[0076] A summary of dimer staining and ELISPOT assay of TILs is shown in FIG.

[0077] Enrichment of dimer-positive TILs Two TIL samples (M37 TIL3 REP1B 2E7 2015-06-03 and M40 TIL3 REP1A 2E7 2015-06-04) were placed in A2 / SSX-2. 41-49 Stained with dimer, A2 / SSX-2 41-49 T cells were purified using flow cytometry-guided sorting (FIG. 29).

[0078] Potential benefits There are many potential advantages of this method. The HLA class I molecules of the present invention may exhibit more native folding and / or protein glycosylation. The HLA molecules of the present invention can be produced in a relatively rapid manner (approximately 2 days versus 4-10 days using conventional methods). Peptides can be exchanged relatively easily in vitro. A simpler protocol resulting in a more native product may also result in significant cost savings. array Soluble A*02:01-wt, nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2)

[0079] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-A*02:01 α1 domain (underlined below) HLA-A*02:01 α2 domain (bold below) HLA-A*02:01 α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO:1: [ka] SEQ ID NO:2: [ka] Soluble A*02:01-Kb, nucleotide sequence (SEQ ID NO:3) and amino acid sequence (SEQ ID NO:4)

[0080] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-A*02:01 α1 domain (underlined below) HLA-A*02:01 α2 domain (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 3 [ka] SEQ ID NO:4 [ka] Soluble A*02:0 1Q115E -K b , nucleotide sequence (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 6)

[0081] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-A*02:01 α1 domain (underlined below) HLA-A*02:01 α2 domain with Q115E mutation (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO:5 [ka] SEQ ID NO:6 [ka] Soluble A*24:02-wt, nucleotide sequence (SEQ ID NO:7) and amino acid sequence (SEQ ID NO:8)

[0082] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-A*24:02 α1 domain (underlined below) HLA-A*24:02 α2 domain (bold below) HLA-A*24:02 α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO:7 [ka] SEQ ID NO:8 [ka] Soluble A*24:02-K b , nucleotide sequence (SEQ ID NO: 9) and amino acid sequence (SEQ ID NO: 10)

[0083] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-A*24:02 α1 domain (underlined below) HLA-A*24:02 α2 domain (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO:9 [ka] SEQ ID NO: 10 [ka] Soluble A*24:02 Q115E -K b , nucleotide sequence (SEQ ID NO: 11) and amino acid sequence (SEQ ID NO: 12)

[0084] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-A*24:02 α1 domain (underlined below) HLA-A*24:02 α2 domain with Q115E mutation (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 11 [ka] SEQ ID NO: 12 [ka] Soluble B*35:01 Q115E -K b , nucleotide sequence (SEQ ID NO: 13) and amino acid sequence (SEQ ID NO: 14)

[0085] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-B*35:01 α1 domain (underlined below) HLA-B*35:01 α2 domain with Q115E mutation (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 13 [ka] [ka] SEQ ID NO: 14 [ka] Soluble B*40:02 Q115E -Kb, nucleotide sequence (SEQ ID NO: 15) and amino acid sequence (SEQ ID NO: 16)

[0086] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-B*40:02 α1 domain (underlined below) HLA-B*40:02 α2 domain with Q115E mutation (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 15 [ka] SEQ ID NO: 16 [ka] Soluble B*44:05 Q115E -Kb, nucleotide sequence (SEQ ID NO: 17) and amino acid sequence (SEQ ID NO: 18)

[0087] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-B*44:05 α1 domain (underlined below) HLA-B*44:05 α2 domain with Q115E mutation (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 17 [ka] SEQ ID NO: 18 [ka] Soluble B*07:02 Q115E -Kb, nucleotide sequence (SEQ ID NO: 19) and amino acid sequence (SEQ ID NO: 20)

[0088] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-B*07:02 α1 domain (underlined below) HLA-B*07:02 α2 domain with Q115E mutation (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 19 [ka] SEQ ID NO: 20 [ka] Soluble B*08:01 Q115E -Kb, nucleotide sequence (SEQ ID NO: 21) and amino acid sequence (SEQ ID NO: 22)

[0089] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-B*08:01 α1 domain (underlined below) HLA-B*08:01 α2 domain with Q115E mutation (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 21 [ka] SEQ ID NO: 22 [ka] Soluble C*05:01 Q115E -Kb, nucleotide sequence (SEQ ID NO: 23) and amino acid sequence (SEQ ID NO: 24)

[0090] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-C*05:01 α1 domain (underlined below) HLA-C*05:01 α2 domain with Q115E mutation (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 23 [ka] SEQ ID NO: 24 [ka] Soluble C*07:01 Q115E-Kb, nucleotide sequence (SEQ ID NO:26) and amino acid sequence (SEQ ID NO:26)

[0091] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-C*07:01 α1 domain (underlined below) HLA-C*07:01 α2 domain with Q115E mutation (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 25 [ka] SEQ ID NO: 26 [ka] Soluble C*07:02 Q115E -Kb, nucleotide sequence (SEQ ID NO: 27) and amino acid sequence (SEQ ID NO: 28)

[0092] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-C*07:02 α1 domain (underlined below) HLA-C*07:02 α2 domain with Q115E mutation (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 27 [ka] SEQ ID NO: 28 [ka] Soluble C*16:01 Q115E -Kb, nucleotide sequence (SEQ ID NO: 29) and amino acid sequence (SEQ ID NO: 30)

[0093] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-C*16:01 α1 domain (underlined below) HLA-C*16:01 α2 domain with Q115E mutation (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 29 [ka] SEQ ID NO: 30 [ka]

[0094] As well as the sequences described above, the present application may also be directed to the following sequences: Soluble B*35:01-wt, nucleotide sequence (SEQ ID NO:31) and amino acid sequence (SEQ ID NO:32)

[0095] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-B*35:01 α1 domain (underlined below) HLA-B*35:01 α2 domain (bold below) HLA-B*35:01 α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 31 [ka] SEQ ID NO: 32 [ka] Soluble B*35:01-Kb, nucleotide sequence (SEQ ID NO:33) and amino acid sequence (SEQ ID NO:34)

[0096] The arrays are enumerated in the following order: Signal peptide from fibroin-L (normal Arial font below) HLA-B*35:01 α1 domain (underlined below) HLA-B*35:01 α2 domain (bold below) Mouse K b α3 domain (italicized below) Flexible GS linker (bold and underlined below) 6xHis tags (bold and italicized below) SEQ ID NO: 33 [ka] SEQ ID NO: 34 [ka]

[0097] While preferred embodiments of the invention are described herein, those skilled in the art will recognize that variations are possible without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the reference list below, are incorporated by reference.

[0098] Prior art documents Non-patent document 1 Wooldridge L,Lissina A,Cole DK,van den Berg HA,Price DA,Sewell AK.Tricks with tetramers:how to get the most from multimeric peptide-MHC.Immunology.2009;126(2):147-164

[0099] Non-patent Document 2 Dolton G,Tungatt K,Lloyd A,Bianchi V,Theaker SM,Trimby A,Holland CJ,Donia M,Godkin AJ,Cole DK,Straten PT,Peakman M,Svane IM,Sewell AK.More tricks with tetramers:a practical guide to staining T cells with peptide-MHC multimers.Immunology.2015;146(1):11-22

[0100] Non-patent Document 3 Rossjohn J,Gras S,Miles JJ,Turner SJ,Godfrey DI,McCluskey J.T cell antigen receptor recognition of antigen-presenting molecules.Annu Rev Immunol.2015;33:169-200

[0101] Non-patent Document 4 Marrack P,Scott-Browne JP,Dai S,Gapin L,Kappler JW.Evolutionarily conserved amino acids that control TCR-MHC interaction.Annu Rev Immunol.2008;26:171-203

[0102] Non-patent Document 5 Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM

[0103] Empty license plate6 Klenerman P, Cerundolo V, Dunbar PR.Tracking T cells with tetramers: new tales from new tools.Nat Rev Immunol.2002;2(4):263-272

[0104] Connection kit7 Janeway C.Immunobiology:the immune system in health and disease (ed 6th).New York:Garland Science;2005

[0105] Empty range8 HLA B*5701 is highly associated with restriction of virus replication in a subgroup of HIV-infected long term nonprogressors.Proc Natl Acad Sci US A.2000;97(6):2709-2714

[0106] Empty range9 Kawase T, Akatsuka Y, Torikai H, Morishima S, Oka A, Tsujimura A, Miyazaki M, Tsujimura K, Miyamura K, Ogawa S, Inoko H, Morishima Y, Kodera Y, Kuzushima K, Takahashi T. Alternative splicing due to an intronic SNP in HMSD generates a novel minor histocompatibility antigen. Blood. 2007;110(3):1055-1063

[0107] Non-patent Document 10 Rodenko B, Toebes M, Hadrup SR, van Esch WJ, Molenaar AM, Schumacher TN, Ovaa H. Generation of peptide-MHC class I complexes through UV-mediated ligand exchange. Nat Protoc. 2006;1(3):1120-1132

[0108] Non-patent Document 11 Bakker AH, Hoppes R, Linnemann C, Toebes M, Rodenko B, Berkers CR, Hadrup SR, van Esch WJ, Heemskerk MH, Ovaa H, Schumacher TN. Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Proc Natl Acad Sci U S A. 2008;105(10):3825-3830

[0109] Non-patent Document 12 Saini SK, Schuster H, Ramnarayan VR, Rammensee HG, Stevanovic S, Springer S. Dipeptides catalyze rapid peptide exchange on MHC class I molecules. Proc Natl Acad Sci U S A. 2015;112(1):202-207

[0110] Non-patent Document 13 Laugel B, van den Berg HA, Gostick E, Cole DK, Wooldridge L, Boulter J, Milicic A, Price DA, Sewell AK. Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties. J Biol Chem. 2007;282(33):23799-23810

[0111] Non-patent Document 14 Nguyen LT, Yen PH, Nie J, Liadis N, Ghazarian D, Al-Habeeb A, Easson A, Leong W, Lipa J, McCready D, Reedijk M, Hogg D, Joshua AM, Quirt I, Messner H, Shaw P, Crump M, Sharon E, Ohashi PS. Expansion and characterization of human melanoma tumor-infiltrating lymphocytes (TILs). PLoS One. 2010;5(11):e13940

[0112] Non-patent Document 15 Kagoya Y, Nakatsugawa M, Yamashita Y, Ochi T, Guo T, Anczurowski M, Saso K, Butler MO, Arrowsmith CH, Hirano N. BET bromodomain inhibition enhances T cell persistence and function in adoptive immunotherapy models. J Clin Invest. 2016;126(9):3479-3494

[0113] Non-patent document 16 Hirano N, Butler MO, Xia Z, Ansen S, Von Bergwelt-Baildon MS, Neuberg D, Freeman GJ, Nadler LM. Engagement of CD83 ligand induces prolonged expansion of CD8+ T cells and preferential enrichment for antigen specificity. Blood. 2006;107(4):1528-1536

[0114] Non-patent document 17 Butler MO, Lee JS, Ansen S, Neuberg D, Hodi FS, Murray AP, Drury L, Berezovskaya A, Mulligan RC, Nadler LM, Hirano N. Long-lived antitumor CD8+ lymphocytes for adoptive therapy generated using an artificial antigen-presenting cell. Clin Cancer Res. 2007;13(6):1857-1867

[0115] Non-patent document 18 Hirano N,Butler MO,Xia Z,Berezovskaya A,Murray AP,Ansen S,Kojima S,Nadler LM.Identification of an immunogenic CD8+ T-cell epitope derived from gamma-globin,a putative tumor-associated antigen for juvenile myelomonocytic leukemia.Blood.2006;108(8):2662-2668

[0116] SPECIFICATIONS19 Imataki O, Ansen S, Tanaka M, Butler MO, Berezovskaya A, Milstein MI, Kuzushima K, Nadler LM, Hirano N. Can IL-21 supplement suboptimal Lck-independent MAPK activation in a STAT-3-dependent manner in human CD8(+) T cells.J Immunol.2012;188(4):1609-1619

[0117] Enclosure 20 Butler MO,Ansen S,Tanaka M,Imataki O,Berezovskaya A,Mooney MM,Metzler G,Milstein MI,Nadler LM,Hirano NA panel of human cell-based artificial APC enables the expansion of long-lived antigen-specific CD4+ T cells restricted by prevalent HLA-DR alleles.Int Immunol.2010;22(11):863-873

[0118] ​​​​21 Wooldridge L, Clement M, Lissina A, Edwards ES, Ladell K, Ekeruche J, Hewitt RE, Laugel B, Gostick E, Cole DK, Debets R, Berrevoets C, Miles JJ, Burrows SR, Price DA, Sewell AK. MHC class I molecules with Superenhanced CD8 binding properties bypass the requirement for cognate TCR recognition and nonspecifically activate CTLs. J Immunol. 2010;184(7):3357-3366

[0119] Non-patent Document 22 Wooldridge L, Lissina A, Vernazza J, Gostick E, Laugel B, Hutchinson SL, Mirza F, Dunbar PR, Boulter JM, Glick M, Cerundolo V, van den Berg HA, Price DA, Sewell AK. Enhanced immunogenicity of CTL antigens through mutation of the CD8 binding MHC class I invariant region. Eur J Immunol. 2007;37(5):1323-1333

[0120] Non-patent Document 23 Hirano N, Butler MO, Von Bergwelt-Baildon MS, Maecker B, Schultze JL, O’Connor KC, Schur PH, Kojima S, Guinan EC, Nadler LM. Autoantibodies frequently detected in patients with aplastic anemia. Blood. 2003;102(13):4567-4575

[0121] Non-Patent Document 24 Hirano N, Butler MO, Xia Z, Berezovskaya A, Murray AP, Ansen S, Nadler LM. Efficient presentation of naturally processed HLA class I peptides by artificial antigen-presenting cells for the generation of effective antitumor responses. Clin Cancer Res. 2006;12(10):2967-2975

[0122] Non-Patent Document 25 Tanaka M, Butler MO, Ansen S, Imataki O, Berezovskaya A, Nadler LM, Hirano N. Induction of HLA-DP4-restricted anti-survivin Th1 and Th2 responses using an artificial antigen-presenting cell. Clin Cancer Res. 2011;17(16):5392-5401

[0123] Non-Patent Document 26 Ochi T, Nakatsugawa M, Chamoto K, Tanaka S, Yamashita Y, Guo T, Fujiwara H, Yasukawa M, Butler MO, Hirano N. Optimization of T-cell Reactivity by Exploiting TCR Chain Centricity for the Purpose of Safe and Effective Antitumor TCR Gene Therapy. Cancer Immunol Res. 2015;3(9):1070-1081

[0124] Non-Patent Document 27 Nakatsugawa M, Yamashita Y, Ochi T, Tanaka S, Chamoto K, Guo T, Butler MO, Hirano N. Specific roles of each TCR hemichain in generating functional chain-centric TCR. J Immunol. 2015;194(7):3487-3500

[0125] Non-Patent Document 28 Feldman SA, Assadipour Y, Kriley I, Goff SL, Rosenberg SA. Adoptive Cell Therapy--Tumor-Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors. Semin Oncol. 2015;42(4):626-639

[0126] Non-Patent Document 29 Robbins PF, Lu YC, El-Gamil M, Li YF, Gross C, Gartner J, Lin JC, Teer JK, Cliften P, Tycksen E, Samuels Y, Rosenberg SA. Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med. 2013;19(6):747-752

[0127] Non-Patent Document 30 Parker KC, Bednarek MA, Coligan JE. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol. 1994;152(1):163-175

Claims

1. 1. A method for producing an HLA class I molecule conjugated to a preselected peptide, comprising: (a) providing an HLA class I molecule of mammalian origin conjugated to a pre-existing peptide; (b) incubating the HLA class I molecule conjugated to the pre-existing peptide with the preselected peptide in vitro; incubating the preselected peptide at a concentration sufficient to displace the existing peptide to produce the HLA class I molecule conjugated to the preselected peptide; The method, wherein the HLA class I molecule comprises α1, α2, α3 and β2m domains.

2. The method of claim 1, wherein the HLA class I molecule is soluble.

3. The method of claim 1 or 2, wherein the HLA class I molecule conjugated to the pre-existing peptide is produced by a mammalian cell transfected with a soluble HLA class I molecule, and the β2m domain may be endogenous or exogenous, preferably exogenous, and is encoded by a second vector.

4. The method of claim 3 , wherein the soluble HLA class I molecule comprises a signal peptide that directs secretion of the HLA class I molecule outside the mammalian cell.

5. The method of claim 4, wherein the soluble HLA class I molecule conjugated to the pre-existing peptide is provided in the culture supernatant of the mammalian cells.

6. The method of any one of claims 1 to 5, wherein the HLA class I molecule is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.

7. The method of claim 6, wherein the HLA class I molecule is HLA-A and comprises the α1, α2 and α3 domains of any of SEQ ID NOs: 2, 4, 6, 8, 10, or 12.

8. The method of claim 6, wherein the HLA class I molecule is HLA-B and comprises the α1, α2 and α3 domains of any of SEQ ID NOs: 14, 16, 18, 20, or 22.

9. The method of claim 6, wherein the HLA class I molecule is HLA-C and comprises the α1, α2 and α3 domains of any of SEQ ID NOs: 24, 26, 28, or 30.

10. The α3 domain of the HLA class I molecule is ｂ The method of claim 6, wherein the α3 domain.

11. The method of claim 6, wherein Gln is substituted with Glu at position 115 in the α2 domain of the HLA class I molecule.

12. 7. The method of claim 6, wherein the HLA class I molecule comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30, and a β2m domain.

13. The method of any one of claims 1 to 12, further comprising multimerizing the HLA class I molecule, preferably into one of a dimer, trimer, tetramer and pentamer.

14. The method of claim 13, wherein the HLA class I molecule is dimerized using an antibody that recognizes a corresponding tag on the HLA class I molecule.

15. 15. The method of claim 14, wherein the tag is a 6xHis tag at the C'-terminus of the α3 domain, preferably linked by a flexible linker such as a GS linker.

16. A kit for producing an HLA class I molecule conjugated to a preselected peptide, comprising an HLA class I molecule derived from a mammal conjugated to an existing peptide and instructions corresponding to the method of claim 1.

17. 17. The kit of claim 16, further comprising the preselected peptide.

18. A polypeptide comprising the α1, α2 and α3 domains of an HLA class I molecule, a signal peptide at the N-terminus, and a 6xHis tag joined by a GS linker at the C-terminus.

19. 19. The polypeptide of claim 18, which is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.

20. A nucleic acid encoding the polypeptide of claim 18 or 19.

21. A vector comprising the nucleic acid of claim 20.

22. A mammalian cell transfected with the vector of claim 21.

23. The mammalian cell of claim 22, further comprising a second vector encoding β2m.

24. A compound comprising a polypeptide according to claim 18 or 19 complexed with a β2m domain.

25. 25. A multimer of at least two compounds according to claim 24.

26. 26. The multimer of claim 25, wherein an antibody that recognizes the 6xHis tag dimerizes the at least two compounds.

27. 1. A method for screening / selecting antigen-specific T cells that recognize a preselected peptide antigen in a T cell population, comprising: providing an HLA class I molecule from a mammal conjugated to said preselected peptide; screening the population of T cells for antigen-specific T cells that bind to an HLA class I molecule from said mammal conjugated to said preselected peptide.

28. first providing an HLA class I molecule from a mammal conjugated to a holder peptide; incubating in vitro the HLA class I molecule conjugated to the holder peptide with the preselected peptide, 28. The method of claim 27, further comprising incubating the preselected peptide at a concentration sufficient to displace the existing peptide to produce the HLA class I molecule conjugated to the preselected peptide.

29. 28. The method of claim 27, wherein the mammalian-derived HLA class I molecule conjugated to the preselected peptide is prepared using the method of any one of claims 1 to 15.

30. The method of any one of claims 27 to 29, wherein the screening comprises flow cytometry.

31. 31. The method of any one of claims 27 to 30, wherein the HLA class I molecule conjugated to the holder peptide comprises any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30, and preferably has a β2m domain.

32. The method of any one of claims 27 to 30, wherein the HLA class I molecule conjugated to the holder peptide comprises a polypeptide of claim 18 or 19, preferably a β2m domain.

33. The method of any one of claims 27 to 32, wherein the antigen-specific T cells that recognize a preselected peptide antigen are tumor-infiltrating lymphocytes.

34. 34. The method of claim 33, wherein the preselected peptide antigen is associated with cancer.

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