Peptide-HLA complexes and methods of producing same
12240886 ยท 2025-03-04
Assignee
Inventors
- Naoto HIRANO (Toronto, CA)
- Munehide NAKATSUGAWA (Sapporo, JP)
- Muhammed Aashiq RAHMAN (Brisbane, AU)
- Kenji MURATA (Toronto, CA)
Cpc classification
C12N5/0638
CHEMISTRY; METALLURGY
C12N5/0639
CHEMISTRY; METALLURGY
A61K40/11
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
A61K47/64
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
Abstract
There is provided herein, the use of mammalian derived HLA class I molecule for in vitro peptide exchange. For example, there is provided a method of producing an HLA class I molecule complexed to a pre-selected peptide 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, wherein 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 1, 2, 3 and 2m domains.
Claims
1. A polypeptide comprising an 1, 2 and 3 domain of an HLA class I molecule, a signal peptide at the N terminus and a 6His tag joined by a GS linker at the C terminus, wherein: (a) the HLA class I molecule is HLA-A and comprises the 1, 2 and 3 domain of any of SEQ ID NOs. 2, 4, 6, 8, 10, and 12; (b) the HLA class I molecule is HLA-B and comprises the 1, 2 and 3 domain of any of SEQ ID NOs. 14, 16, 18, 20, and 22; (c) the HLA class I molecule is HLA-C and comprises the 1, 2 and 3 domain of any of SEQ ID NOs. 24, 26, 28, and 30; or (d) the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.
2. A compound comprising the polypeptide of claim 1 complexed with a 2m domain.
3. A multimer of at least two compounds according to claim 2.
4. The multimer of claim 3, wherein an antibody recognizing the 6His tag dimerizes the at least two compounds.
5. The polypeptide of claim 1, which is soluble.
6. The compound of claim 2, wherein the 2m domain is exogenous.
7. The polypeptide of claim 1, wherein the HLA class I molecule is HLA-A and comprises the 1, 2 and 3 domains of any of SEQ ID NOs. 2, 4, 6, 8, 10, or 12.
8. The polypeptide of claim 1, wherein the HLA class I molecule is HLA-B and comprises the 1, 2 and 3 domains of any of SEQ ID NOs. 14, 16, 18, 20, or 22.
9. The polypeptide of claim 1, wherein the HLA class I molecule is HLA-C and comprises the 1, 2 and 3 domains of any of SEQ ID NOs. 24, 26, 28, or 30.
10. The polypeptide of claim 1, wherein the 3 domain of the HLA class I molecule is the mouse K.sup.b 3 domain.
11. The polypeptide of claim 1, wherein in the 2 domain of the HLA class I molecule, Gln has been replaced with Glu at position 115.
12. The compound of claim 2, wherein the HLA class I molecule comprises the amino acid sequence set forth in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.
13. The multimer of claim 3, wherein the multimer is a dimer, trimer, tetramer, or pentamer.
14. The compound of claim 2, wherein the HLA class I molecule is complexed to a pre-selected peptide.
15. The compound of claim 14, wherein the pre-selected peptide comprises a portion of a polypeptide selected from the group consisting of a WT1 polypeptide, a MIA polypeptide, an ALX1 polypeptide, a GAPDHS polypeptide, an S100B polypeptide, an ABC5 polypeptide, an EXTL1 polypeptide, a CPN1 polypeptide, a TSPAN10 polypeptide, a GJB1 polypeptide, an MITF polypeptide, a DUSP4 polypeptide, a cyclin-A1 polypeptide, an HERV-K-MEL polypeptide, an LAGE-1 polypeptide, a MAGE polypeptide, an LAGE-2 polypeptide, an SSX-2 polypeptide, an XAGE-1b polypeptide, a CEA polypeptide, a gp100 polypeptide, an NY-BR-1 polypeptide, a TRP-2 polypeptide, a tyrosinase polypeptide, a CD274 polypeptide, a CPSF polypeptide, a cyclin D1 polypeptide, an IDO1 polypeptide, an mdm-2 polypeptide, a p53 polypeptide, a PRAME polypeptide, a SOX10 polypeptide, a survivin polypeptide, a telomerase polypeptide, a wild-type MART1 polypeptide, a heteroclitic MART1 polypeptide, a wild-type NY-ESO-1 polypeptide, a heteroclitic NY-ESO-1 polypeptide, an HIV pol polypeptide, and an HTLV-1 tax polypeptide.
16. The compound of claim 14, wherein the pre-selected peptide comprises an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 35-122.
Description
BRIEF DESCRIPTION OF FIGURES
(1) Embodiments of the invention may best be understood by referring to the following description and accompanying drawings. In the drawings:
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DETAILED DESCRIPTION
(31) We have developed a novel technology which enables high throughput production of mammalian-derived peptide/HLA class I (pHLA) multimers that can stain low affinity TCRs. One example application of this technology is the generation of personalized pHLA reagents which enables high-throughput measurement of antitumor T cell responses in cancer patients.
(32) According to one aspect, there is provided a method of producing an HLA class I molecule complexed to a pre-selected peptide by providing a mammalian derived HLA class I molecule complexed first to an existing peptide. The HLA class I molecule complexed to the existing peptide is then incubated, in vitro, with the pre-selected peptide at a concentration sufficient to replace the existing peptide, thereby producing the HLA class I molecule complexed to the pre-selected peptide. The HLA class I molecule comprises 1, 2, 3 and 2m domains. In some embodiments, the HLA class I molecule is soluble.
(33) Human Leukocyte Antigen
(34) The HLA system is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. These cell-surface proteins are responsible for the regulation of the immune system in humans. HLA genes are highly polymorphic, and different classes have different functions. HLA class I genes encoding MHC class I molecules function to display or present peptide fragments of non-self or self proteins from within the cell to cytotoxic T cells.
(35) As used herein, the expression HLA class I molecule refers to a protein molecule derived from the expression of wild type or variant HLA class I genes encoding MHC class I molecules. A schematic representation of the general structure of an HLA class I molecule, including its 1, 2, 3 and 2m domains, is depicted in
(36) The schematic representation also illustrates a peptide complexed to the HLA class I molecule. As used herein, the expression peptide refers to peptide fragments that are capable of complexing with the HLA class I molecule and are displayed or presented by the HLA class I molecule. Such peptides have been well described in the art. In general, these particular peptides are about 8-15 amino acids in length but can also vary from between 8-10, 7-11, or 6-12 amino acids in length.
(37) For some HLA class I genes, pHLA production using bacteria is difficult partly because of poor refolding. Furthermore, bacterially expressed and in vitro refolded pMHC proteins may not have exactly the same higher structure as those produced in mammalians and refolded in vivo. As used herein, the expression mammalian derived refers to production of molecules utilizing mammalian cell systems which are well known in the art, such as human cell lines (for example, Hela, HEK293, HEK293T and their derivatives), monkey cell lines (for example, CV-1, COS and their derivatives), mouse cell line (for example, NIH3T3 and their derivatives, NS-1 and their derivatives), hamster cell lines (for example, BHK, CHO and their derivatives). In one embodiment, human cell lines are used. In one example, HEK 293T cell lines can be used. The HLA class I molecule complexed to the existing peptide is produced by a mammalian cell transfected with a soluble HLA class I molecule, wherein the 2m domain may be endogenous or exogenous. In preferred embodiments, the 2m domain is exogenous and encoded on a second vector.
(38) In some embodiments, the soluble HLA class I molecule comprises a signal peptide directing secretion of the HLA class I molecule outside of the mammalian cell. In other embodiments, the soluble HLA class I molecule complexed to the existing peptide is provided in the supernatant of a culture of the mammalian cells.
(39) HLA Class I Genes
(40) The HLA class I genes is a family of genes. The HLA class I molecule can be HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.
(41) As used herein, HLA-A refers to a protein molecule derived from the expression of an HLA-A gene. HLA-B refers to a protein molecule derived from the expression of an HLA-B gene. HLA-C refers to a protein molecule derived from the expression of an HLA-C gene. HLA-D refers to a protein molecule derived from the expression of an HLA-D gene. HLA-E refers to a protein molecule derived from the expression of an HLA-E gene. HLA-F refers to a protein molecule derived from the expression of an HLA-F gene. HLA-G refers to a protein molecule derived from the expression of an HLA-G gene. All of the genes HLA-A to HLA-G are part of the HLA class I family of genes.
(42) Amino Acid Sequences of the HLA Class I Molecule
(43) The HLA class I molecule may have a number of amino acid sequence variants.
(44) In some embodiments, the 3 domain of the HLA class I molecule is the mouse Kb 3 domain (designated Kb)). In other embodiments, in the 2 domain of the HLA class I molecule, Gln has been replaced with Glu at position 115 (designated Q115E).
(45) Exemplary HLA class I molecules include but are not limited to the following.
(46) The HLA class I molecule may be HLA-A and comprises the 1, 2 and 3 domains of any of SEQ ID NOs. 6 or 12. In other embodiments, the HLA-A 1, 2 and 3 domains may be wildtype as in SEQ ID NOs. 2 or 14 respectively. Additionally, in yet other embodiments, the 1 and 2 domains are wildtype and the 3 domain of the HLA class I molecule is the mouse Kb 3 domain as in SEQ ID NOs. 4 or 10 respectively. Any combination of the foregoing is also possible.
(47) The HLA class I molecule can be HLA-B and comprises the 1, 2 and 3 domains of any of SEQ ID NOs. 14, 16, 18, 20, or 22. As with exemplary HLA-A molecules, the 1, 2 and 3 domains may be wildtype, or be select variants, such as Kb and Q115E, or any combinations thereof.
(48) The HLA class I molecule can be HLA-C and comprises the 1, 2 and 3 domains of any of SEQ ID NOs. 24, 26, 28, or 30. As with exemplary HLA-A molecules, the 1, 2 and 3 domains may be wildtype, or be select variants, such as Kb and Q115E, or any combinations thereof.
(49) In yet other embodiments, the HLA class I molecule comprises the 1, 2 and 3 domains described herein with a 2m domain.
(50) Multimers
(51) The HLA class I molecule may also be multimerized. According to a further aspect, the method described above further comprises multimerizing the HLA class I molecules, preferably into one of dimers, trimers, tetramers and pentamers.
(52) In some embodiments, the HLA class I molecules are dimerized using an antibody that recognizes a corresponding tag on HLA class I molecule. In further embodiments, the tag is a 6His tag at the C end of the 3 domain, preferably connected by a flexible linker, more preferably a GS linker. Other suitable tags for antibody binding are known in the art. Examples of acceptable tags are numerous and include AviTag, Calmodulin-tag, polyglutamate tag, His-tag, Myc-tag, and VSV-tag. Examples of acceptable flexible linkers are numerous; see for example Chen et al, Adv Drug Deliv Rev. 2013 Oct. 15; 65 (10): 1357-1369.
(53) Kits and Reagents
(54) According to a further aspect, there is provided a kit for producing an HLA class I molecule complexed to a pre-selected peptide, comprising a mammalian derived HLA class I molecule complexed to an existing peptide and instructions corresponding to the method described above. In some embodiments, the kit further comprises the pre-selected peptide.
(55) According to a further aspect, there is provided a polypeptide comprising the 1, 2 and 3 domain of an HLA class I molecule, a signal peptide at the N terminus and a 6His tag joined by a GS linker at the C terminus. In some embodiments, the polypeptide is SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.
(56) According to a further aspect, there is provided a nucleic acid encoding the polypeptide described above.
(57) According to a further aspect, there is provided a vector comprising the nucleic acid described above.
(58) According to a further aspect, there is provided a mammalian cell transfected with the vector described above. In some embodiments, the mammalian cell further comprises a second vector encoding 2m.
(59) According to a further aspect, there is provided a compound comprising the polypeptide described above complexed with a 2m domain.
(60) According to a further aspect, there is provided a multimer of at least two of the compounds described above. In some embodiments, the at least two compounds are dimerized by an antibody recognizing the 6His tag.
(61) The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
(62) T-Cell Screening and Selection, Including Tumor-Infiltrating Lymphocytes
(63) It is known that HLA class I molecules complexed to a pre-selected peptide can be used to screen/select for T-cells that recognize said peptide antigen through its T-cell receptor. Advantageously, the mammalian derived HLA class I molecules described herein allow the skilled person to swap out an existing (or holder) peptide with a pre-selected peptide of interest. This was not possible with existing bacteria-derived HLA class I molecules. Rather, the existing bacteria-derived HLA class I molecules had to be produced, denatured and then re-folded with the peptide antigen of interest.
(64) The present mammalian-derived HLA class I molecules therefore represent a streamlined and more flexible procedure to easily produce molecules that can present a peptide antigen. For example, the present mammalian-derived HLA class I molecules can be pre-made, the holder peptide being swapped before use. Further, the present mammalian-derived HLA class I molecules are likely more representative of a natural HLA class I molecules as they do not have to be refolded and are glycosylated.
(65) Accordingly, in an aspect, the method of screening/selecting in a population of T-cells for antigen specific T-cells that recognize pre-selected peptide antigens, the method comprising: providing a mammalian-derived HLA class I molecule complexed to the pre-selected peptides; screening the population of T-cells for antigen specific T-cells that bind the mammalian-derived HLA class I molecule complexed to the pre-selected peptides.
(66) In some embodiments, the method further comprises first providing a mammalian-derived HLA class I molecule complexed to a holder peptide; incubating, in vitro, the HLA class I molecule complexed to the holder peptide with the pre-selected peptide, wherein 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.
(67) In some embodiments, the mammalian-derived HLA class I molecule complexed to the pre-selected peptide is prepared using the method of described herein.
(68) In some embodiments, the screening comprises flow cytometry.
(69) In some embodiments, the HLA class I molecule complexed to the holder peptide comprises any one of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30, preferably with a 2m domain.
(70) In some embodiments, the HLA class I molecule complexed to the holder peptide comprises the polypeptide described herein, preferably with a 2m domain.
(71) In some embodiments, the method may be used to screen/select for T-Cell populations associated with a cancer. Cancer may include adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain/cns tumors, breast cancer, castleman disease, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (gist), gestational trophoblastic disease, hodgkin disease, kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia (acute lymphocytic, acute myeloid, chronic lymphocytic, chronic myeloid, chronic myelomonocytic), liver cancer, lung cancer (non-small cell, small cell, lung carcinoid tumor), lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin 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 macroglobulinemia, or wilms tumor.
(72) In some embodiments, the antigen specific T-cells that recognize pre-selected peptide antigens are tumor-infiltrating lymphocytes.
(73) In some embodiments, the pre-selected peptide antigens are associated with cancer.
Examples
Materials and Methods
(74) Peptides
(75) Synthetic peptides were purchased from ProImmune, Genway Biotech, and GenScript. Peptides used were A2-restricted heteroclitic MART1.sub.26-35 (ELAGIGILTV), heteroclitic NY-ESO-1.sub.157-165 (SLLMWITQV), A24-restricted heteroclitic WT1.sub.235-243 (CYTWNQMNL), B35-restricted wild-type EBNA-1.sub.407-417 (HPVGEADYFEY) peptides, B44-restricted wild-type EBNA-6.sub.281-290 (EENLLDFVRF), C7-restricted wild-type MAGE-A1.sub.289-297 (RVRFFFPSL), and C7-restricted wild-type MAGE-A 12.sub.170-178 (VRIGHLYIL) peptides. A2 peptides used to stain TILs are listed in Table 1 below.
(76) TABLE-US-00001 TABLE1 A2peptidestested SEQ ID Name Sequence NO. 1 WT1(37-) VLDFAPPGA 35 2 WT1(126-) RMFPNAPYL 36 3 WT1(87-) SLGEQQYSV 37 4 WT1(235-) CMTWNQMNL 38 5 MIA(54-) YMAPDCRFL 39 6 MIA(99-) RLGYFPSSI 40 7 ALX1(142-) LQLEELEKV 41 8 ALX1(170-) ELTEARVQV 42 9 GAPDHS(358-) FLGDTHSSI 43 10 GAPDH2(345-) ILAYTEDEV 44 11 S100B(44-) FLEEIKEQEV 45 12 S100B(74-) FMAFVAMVT 46 13 ABCB5(1078-) LLDEATSAL 47 14 ABCB5(700-) VLNGTVHPV 48 15 EXTL1(249-) VLLSPRWEL 49 16 EXTL1(13-) FLWDAYFSS 50 17 EXTL1(330-) WLALSASWL 51 18 CPN1(379-) LLLPGIYTV 52 19 CPN1(249-) KLFQKLAKV 53 20 CPN1(297-) YLHTNCFEI 54 21 TSPAN10(81-) FLSNFPFSL 55 22 TSPAN10(94-) ALAIGLWGL 56 23 TSPAN10(142-) ALCENTCLL 57 24 GJB1(155-) LLYPGYAMV 58 25 GJB1(5-) GLYTLLSGV 59 26 GJB1(147-) AVFMYVFYL 60 27 MITF(378-) LMDDTLSPV 61 28 MITF(142-) LQMANTLPV 62 29 MITF(392-) LLSSVSPGA 63 30 DUSP4(362-) SQFVFSFPV 64 31 DUSP4(326-) QLLQFESQV 65 32 DUSP4(53-) FLAHSAGYI 66 33 cyclin-A1(227-) FLDRFLSCM 67 34 cyclin-A1(341-) SLIAAAAFCLA 68 35 HERV-K-MEL(1-) MLAVISCAV 69 36 LAGE-1(1-) MLMAQEALAFL 70 37 MAGE-A1(278-) KVLEYVIKV 71 38 MAGE-A2(157-) YLQLVFGIEV 72 39 MAGE-A3(271-) FLWGPRALV 73 40 MAGE-A3(112-) KVAELVHFL 74 41 MAGE-A4(230-) GVYDGREHTV 75 42 MAGE-A9(223-) ALSVMGVYV 76 43 MAGE-A10(254-) GLYDGMEHL 77 44 MAGE-A12(271-) FLWGPRALV 78 45 MAGE-C1(959-) ILFGISLREV 79 46 MAGE-C1(1083-) KVVEFLAML 80 47 LAGE-2(1-) MLMAQEALAFL 81 48 SSX-2(41-) KASEKIFYV 82 49 XAGE-1b(21-) RQKKIRIQL 83 50 CEA(691-) IMIGVLVGV 84 51 gp100(154-) KTWGQYWQV 85 52 gp100(177-) AMLGTHTMEV 86 53 gp100(178-) MLGTHTMEV 87 54 gp100(209-) ITDQVPFSV 88 55 gp100(280-) YLEPGPVTA 89 56 gp100(457-) LLDGTATLRL 90 57 gp100(476-) VLYRYGSFSV 91 58 gp100(570-) SLADTNSLAV 92 59 gp100(619-) RLMKQDFSV 93 60 gp100(639-) RLPRIFCSC 94 61 NY-BR-1(904-) SLSKILDTV 95 62 TRP-2(180-) SVYDFFVWL 96 63 TRP-2(360-) TLDSQVMSL 97 64 tyrosinase(1-) MLLAVLYCL 98 65 tyrosinase(8-) CLLWSFQTSA 99 66 tyrosinase(369-) YMDGTMSQV 100 67 CD274(15-) LLNAFTVTV 101 68 CPSF(250-) KVHPVIWSL 102 69 CPSF(1360-) LMLQNALTTM 103 70 cyclinD1(101-) LLGATCMFV 104 71 IDO1(199-) ALLEIASCL 105 72 mdm-2(53-) VLFYLGQY 106 73 p53(264-) LLGRNSFEV 107 74 p53(65-) RMPEAAPPV 108 75 PRAME(100-) VLDGLDVLL 109 76 PRAME(142-) SLYSFPEPEA 110 77 PRAME(300-) ALYVDSLFFL 111 78 PRAME(425-) SLLQHLIGL 112 79 SOX10(332-) AWISKPPGV 113 80 SOX10(331-) SAWISKPPGV 114 81 survivin(95-) ELTLGEFLKL 115 82 Telomerase(865-) RLVDDFLLV 116 83 WildtypeMART1 AAGIGILTV 117 (27-35) 84 HeterocliticMART1 ELAGIGILTV 118 (26-35) 85 WildtypeNY-ESO-1 SLLMWITQC 119 (157-165) 86 HeterocliticNY-ESO-1 SLLMWITQV 120 (157-165) 87 HIVpol(476-484) ILKEPVHGV 121 88 HTLV-1tax(11-19) LLFGYPVYV 122 89 Nopeptideexchange
Cells and cDNAs
(77) HEK293T cells were obtained from American Type Culture Collection. TILs isolated from an HLA-A2.sup.+ patient with metastatic melanoma were grown in vitro as reported previously.sup.14. Appropriate informed consent and institutional review board approval were obtained. All clonotypic TCR genes were reconstituted in Jurkat 76/CD8 cells or primary T cells as previously described. cDNAs were fused with puromycin resistance gene via internal ribosome entry site.sup.15,16. Transduced cells were isolated by puromycin selection. All cDNAs were cloned into pMX vector and transduced using 293GPG cell-based retrovirus system.sup.16-19.
(78) Flow Cytometry Analysis
(79) mAbs recognizing the following surface antigens were used: 2m (551337, BD BioSciences), His (ab72467, Abcam). Mouse isotype controls were from BD BioSciences. Surface molecular staining was carried out as described elsewhere.sup.16,20.
(80) Immunoblotting
(81) For immunoblotting, cells were extracted in 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 -glycerophosphate, 1% NP-40, 1 mM PMSF, and 1 g/ml Aprotinin). Cell extracts were centrifuged at 10,000 g for 10 min at 4 C. and separated by Tris-Glycine SDS-PAGE followed by electrophoretic transfer to Immobilon-P membrane (Millipore). After blocking with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with the indicated mouse anti-His mAb (sc-53073, Santa Cruz Biotechnology) at 4 C. overnight, washed and incubated with HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Promega) at room temperature for 1 hr. The signal was detected by enhanced chemiluminescence (GE Healthcare).
(82) Results and Discussion
(83) Structure of Soluble Monomeric Peptide/HLA Class I (pHLA) Complexes
(84) HLA class I molecules are heterodimers consisting of two polypeptide chains, and 2-microglobulin (2m), which are non-covalently linked. While the chain is highly polymorphic, the 2m subunit is monomorphic. The HLA class I 1 and 2 domains constitute a groove for peptides of 8-10 amino acids in length. The 3 domain, which contains a transmembrane domain, binds 2m. While TCR on the surface of cytotoxic T cells recognizes the peptides presented by the HLA class I 1 and 2 domains to check antigenicity, the CD8 co-receptor binds the 2 and 3 domains to stabilize the interaction between the TCR and pHLA. Therefore, enhancement of the CD8 and HLA class I interaction leads to the improvement in the strength of the interaction between pHLA and cognate TCR.
(85) It has been demonstrated that replacement of HLA class I 3 domain with mouse K.sup.b 3 domain, named hereafter class I-K.sup.b, enhances the interaction between the class I and CD8 by 10 times. Substitution of the Gln (Q) residue at position 115 of the 2 domain with a Glu (E) residue, named hereafter class I.sup.Q115E, further improves the interaction by 1.5 times.sup.21,22. By fusing the extracellular domain of wild-type (wt) HLA class I with a Gly-Ser (GS) flexible linker followed by a 6His tag, we have generated soluble class I-wt. Soluble class I-K.sup.b and class I.sup.Q115E_K.sup.b were similarly produced. Nucleotide and amino acid sequences of soluble class I-wt, class I-K.sup.b, and class I.sup.Q115E-K.sup.b genes used in this study are listed below.
(86) Production of Soluble Monomeric pHLA Complexes Using Mammalian Cells
(87) HEK293T cells were initially transfected with 2m gene and subsequently with soluble HLA class I-K.sup.b or HLA class I.sup.Q115E_K.sup.b gene using the pMX vector and 293GPG cell-based retrovirus system 16-19.
(88) Enhanced 2m Expression by Gene Transduction
(89) Flow cytometry analysis following 2m-specific mAb staining demonstrated enhanced 2m expression in HEK293T cells stably transfected with 2m gene along with a soluble form of HLA-A2-K.sup.b or A2.sup.Q115E_K.sup.b. HLA-A*02:01 (A2) gene, which is one of the most frequent HLA class I alleles, was used as a representative HLA class I gene. The same strategy was applied to generate HEK293T-derived cell lines stably expressing a soluble form of other class I genes.
(90) Cellular Expression of Soluble Monomeric Peptide/HLA (pHLA) in HEK293T Transfectants
(91) Total cell lysates of HEK293T cells stably expressing soluble HLA-A2-K.sup.b or A2.sup.Q115E_K.sup.b gene in conjunction with or without 2m gene were blotted with anti-His mAb as reported previously.sup.23-25 Cellular expression of soluble HLA-A2-K.sup.b and A2.sup.Q115E_K.sup.b was demonstrated at the protein level.
(92) Secretion of Soluble Monomeric pHLA Complexes into the Supernatant.
(93) Supernatant of HEK293T cells transfected with soluble HLA-A2-K.sup.b or A2.sup.Q115E_K.sup.b gene along with or without 2m gene was harvested and blotted with His-specific mAb. Indicated amounts of bacterially-expressed and 6His-tagged HLA-A2/heteroclitic MART1.sub.26-35 monomer (NIH tetramer core facility) were loaded as controls. Ten l of each supernatant was loaded per lane without any concentration. Secretion of monomeric HLA-A2-K.sup.b and A2.sup.Q115E_K.sup.b into the medium was confirmed.
(94) Monomeric pHLA Complexes were Secreted Only when 2m was Overexpressed.
(95) When HEK293T cells were transduced with soluble HLA-A2-K.sup.b or A2.sup.Q115E_K.sup.b gene alone without 2m gene, secretion of soluble A2-K.sup.b and A2.sup.Q115E_K.sup.b into the medium was not detectable. This suggests that the endogenous 2m expression level was not sufficient to enable the secretion of ectopically expressed soluble A2-K.sup.b and A2.sup.Q115E_K.sup.b.
(96) Production of Monomeric pHLA Loaded with Peptide of Interest by In Vitro Peptide Exchange.
(97) Soluble HLA-A2-K.sup.b and A2.sup.Q115E_K.sup.b-containing supernatant produced by the HEK293T transfectants were simply mixed with the indicated concentration of A2-restricted peptide of interest at room temperature for in vitro peptide exchange (see
(98) Dimerization of Monomeric pHLA Complexes.
(99) Soluble HLA classI.sup.Q115E_K.sup.b monomer in the HEK293T conditioned medium was dimerized using anti-His mAb conjugated with fluorochrome such as phycoerythrin (PE) at at 2:1 molar ratio. Note that the soluble proteins were fused with a 6His tag at the C-terminus.
(100) Overall Protocol for Production of Dimeric pHLA Complexes to Stain Antigen-Specific T Cells.
(101) Stable HEK293T cell lines ectopically expressing soluble monomeric class I.sup.Q115E-K.sup.b and 2m were established as described above. The stable cell lines were grown until confluent and medium was changed. After 48 hrs, the conditioned medium was harvested and immediately used or frozen until use. The supernatant was loaded with class I-restricted peptide of interest for 24 hrs at 37 C. for in vitro peptide exchange. The soluble monomeric class I.sup.Q115E_K.sup.b loaded with the peptide was dimerized using fluorochrome-conjugated anti-His mAb for 24 hrs at 4 C. (see
(102) Peptide Exchange Occurs in the Supernatant by Simple Mixing.
(103) Soluble A2.sup.Q115E_K.sup.b monomer was loaded with A2/MART1.sub.26-35 (ELAGIGILTV) or A2/NY-ESO-1.sub.157-165 (SLLMWITQV) peptide by simple mixing, dimerized with PE-conjugated anti-His mAb, and used to stain human Jurkat 76/CD8 T cells expressing clonotypic cognate TCR (see
(104) Soluble Monomeric A2.sup.Q115E_K.sup.b Stains High Avidity Antigen-Specific T Cells.
(105) Soluble A2.sup.Q115E_K.sup.b monomer was loaded with A2/MART1.sub.26-35 or A2/NY-ESO-1.sub.157-165 peptide by simple mixing and, without dimerization, directly used to stain Jurkat 76/CD8 T cells expressing clonotypic cognate TCR. Jurkat 76/CD8 cells expressing high but not low affinity TCRs were stained by monomeric soluble A2.sup.Q115E-K.sup.b loaded with cognate peptide (see
(106) Soluble Dimeric A2.sup.Q115E-K.sup.b Stains Both High and Low Avidity Antigen-Specific T Cells.
(107) Soluble monomeric A2.sup.Q115E-K.sup.b containing supernatant was loaded with A2/MART1.sub.26-35 or A2/NY-ESO-1.sub.157-165 peptide by simple mixing, dimerized with PE-conjugated anti-His mAb, and utilized to stain Jurkat 76/CD8 T cells expressing clonotypic cognate TCR (see
(108) Soluble Dimeric Class I.sup.Q115E-K.sup.b Stains Low Affinity TCRs Better than Pentamer (ProImmune) or Tetramer (NIH)
(109) PE-conjugated soluble dimeric A2.sup.Q115E_K.sup.b and A24.sup.Q115E_K.sup.b were loaded with A2/MART1.sub.26-35 and A24/WT1.sub.235-243 (CYTWNQMNL) peptides, respectively. The loaded dimers were employed to stain Jurkat 76/CD8 T cells expressing clonotypic cognate TCRs with various affinities.sup.26,27. Our dimer stained low affinity TCRs better than Pentamer (ProImmune) and NIH's tetramer (see
(110) Soluble Dimeric HLA-B.sup.Q115E_K.sup.b Works as Well.
(111) Soluble monomeric HLA-B35.sup.Q115E-K.sup.b was loaded with B35/EBNA-1.sub.407-417 (HPVGEADYFEY) peptide, dimerized with PE-conjugated anti-His mAb, and used to stain Jurkat 76/CD8 T cells expressing clonotypic cognate TCR (see
(112) Soluble monomeric HLA-B44.sup.Q115E_K.sup.b was loaded with B44/EBNA-6.sub.281-290 (EENLLDFVRF), dimerized with PE-conjugated anti-His mAb, and used to stain Jurkat 76/CD8 T cells expressing clonotypic cognate TCR (
(113) Soluble Dimeric HLA-C.sup.Q115E_K.sup.b Works as Well.
(114) Soluble monomeric HLA-C7.sup.Q115E_K.sup.b was loaded with C7/MAGE-A1.sub.289-297 (RVRFFFPSL) peptide and C7/MAGE-A12.sub.170-178 (VRIGHLYIL) peptide, dimerized with PE-conjugated anti-His mAb, and used to stain Jurkat 76/CD8 T cells expressing clonotypic cognate TCR (
(115) Staining of In Vitro Expanded Tumor-Infiltrating Lymphocytes with a Panel of Soluble A2 Dimers.
(116) Peripheral T cells do not always reflect the immune response to the tumor taking place in cancer patients and antitumor cellular immunity in the periphery does not often correlate with prognosis. In contrast, tumor infiltrating lymphocytes (TILs) interact more closely with the tumor cells and are likely to reflect the tumor host interaction with higher fidelity. The use of TILs as a graft for adoptive cell transfer therapy to treat cancer has been pioneered by Rosenberg's group at the National Cancer Institute in the US.sup.28.
(117) It is believed that TILs are a polyclonal population of T cells with various antigen specificities.sup.29. To investigate the tumor specificity of TILs using our soluble dimer pHLA technology, TILs were isolated from nine HLA-A2.sup.+ patients with metastatic melanoma and grown in vitro as reported previously.sup.14. A large panel of 8-11 mer peptides derived from proteins highly expressed by autologous tumor cells were predicted using publicly available algorithms as reported previously (see Table 1).sup.18,23,30. A library of soluble dimeric A2.sup.Q115E_K.sup.b loaded with the predicted A2 peptides were produced as described above and used to stain the TILs (see
(118) Functional Assays of Dimer.sup.+ T Cells
(119) Using ELISPOT assays, A2-restricted peptide-specific IFN- secretion was confirmed for all the 6 TIL samples for which dimer staining was positive. PVDF plates (Millipore, Bedford, MA) were coated with capture mAb (1D1K; MABTECH, Mariemont, OH). TILs were incubated with 210.sup.4 per well of T2 cells in the presence of each peptide for 20-24 hours at 37 C. The plates were washed and incubated with biotin-conjugated detection mAb (7-B6-1; MABTECH). HRP-conjugated SA (Jackson ImmunoResearch) was then added, and IFN- spots were developed. The reaction was stopped by rinsing thoroughly 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) (
(120) A Summary of Dimer Staining and ELISPOT Assays of TILs is Shown in
(121) Two TIL samples (M37 TIL3 REP1B 2E7 2015 Jun. 3 and M40 TIL3 REP1A 2E7 2015 Jun. 4) were stained with A2/SSX-2.sub.41-49 dimer and A2/SSX-2.sub.41-49 T cells were purified using flow-cytometry-guided sorting (
(122) Possible Advantages.
(123) There are a number of possible advantages of the present methods. The present HLA class I molecules may represent a more natural folding and/or glycosylation of the protein. The present HLA molecules might be produced in relatively quick fashion ( 2 days vs. 4-10 days using conventional methods). Peptides might be exchanged relatively simply in vitro. A simpler protocol resulting in a more natural product might also result in significant cost savings.
(124) Sequences
(125) Soluble A*02:01-Wt. Nucleotide Sequence (SEQ ID NO. 1) and Amino Acid Sequence (SEQ ID NO. 2)
(126) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-A*02:01 1 domain (underlined below) HLA-A*02:01 2 domain (in bold below) HLA-A*02:01 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(127) TABLE-US-00002 SEQIDNO.1: ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGAAGCCACAGCATGCGGTACTTTTTCACCAGCGTGTCCAGACCCGG CAGAGGCGAGCCCAGATTCATTGCCGTGGGCTACGTGGACGACACCCAG TTCGTCAGATTCGACAGCGACGCCGCCAGCCAGCGGATGGAACCTAGAG CCCCTTGGATCGAGCAGGAAGGCCCCGAGTACTGGGACGGCGAGACACG GAAAGTGAAGGCCCACAGCCAGACCCACAGAGTGGATCTGGGCACCCTG CGGGGCTACTACAATCAGTCTGAGGCCGGCTCCCACACCGTGCAGAGGA TGTACGGCTGTGACGTGGGCAGCGACTGGCGGTTCCTGAGAGGCTACCA CCAGTACGCCTACGACGGCAAGGACTATATCGCCCTGAAAGAGGACCTG CGGAGCTGGACAGCCGCCGATATGGCCGCCCAGACCACCAAGCACAAAT GGGAAGCCGCCCACGTGGCCGAGCAGCTGAGAGCTTATCTGGAAGGCAC CTGTGTGGAATGGCTGCGGAGATACCTGGAAAACGGCAAAGAGACACTG CAGCGCACGGACGCCCCCAAAACGCATATGACTCACCACGCTGTCTCTG ACCATGAAGCCACCCTGAGGTGCTGGGCCCTGAGCTTCTACCCTGCGGA GATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACG GAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CGGCTGTGGTGGTGCCTTCTGGACAGGAGCAGAGATACACCTGCCATGT GCAGCATGAGGGTTTGCCCAAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.2: MMRPIVLVLLFATSALAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQ FVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTL RGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDL RSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETL QRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEFTLTWQRDGEDQTQDT ELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEFG S
Z
Soluble A*02:01-K.sup.b, Nucleotide Sequence (SEQ ID NO. 3) and Amino Acid Sequence (SEQ ID NO. 4)
(128) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-A*02:01 1 domain (underlined below) HLA-A*02:01 2 domain (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(129) TABLE-US-00003 SEQIDNO.3 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGAAGCCACAGCATGCGGTACTTTTTCACCAGCGTGTCCAGACCCGG CAGAGGCGAGCCCAGATTCATTGCCGTGGGCTACGTGGACGACACCCAG TTCGTCAGATTCGACAGCGACGCCGCCAGCCAGCGGATGGAACCTAGAG CCCCTTGGATCGAGCAGGAAGGCCCCGAGTACTGGGACGGCGAGACACG GAAAGTGAAGGCCCACAGCCAGACCCACAGAGTGGATCTGGGCACCCTG CGGGGCTACTACAATCAGTCTGAGGCCGGCTCCCACACCGTGCAGAGGA TGTACGGCTGTGACGTGGGCAGCGACTGGCGGTTCCTGAGAGGCTACCA CCAGTACGCCTACGACGGCAAGGACTATATCGCCCTGAAAGAGGACCTG CGGAGCTGGACAGCCGCCGATATGGCCGCCCAGACCACCAAGCACAAAT GGGAAGCCGCCCACGTGGCCGAGCAGCTGAGAGCTTATCTGGAAGGCAC CTGTGTGGAATGGCTGCGGAGATACCTGGAAAACGGCAAAGAGACACTG CAGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.4 MMRPIVLVLLFATSALAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQ FVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTL RGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDL RSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPG S
Z
Soluble A*02:01.sup.Q115E-K.sup.b. Nucleotide Sequence (SEQ ID NO. 5) and Amino Acid Sequence (SEQ ID NO. 6)
(130) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-A*02:01 1 domain (underlined below) HLA-A*02:01 2 domain with Q115E mutation (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(131) TABLE-US-00004 SEQIDNO.5 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGAAGCCACAGCATGCGGTACTTTTTCACCAGCGTGTCCAGACCCGG CAGAGGCGAGCCCAGATTCATTGCCGTGGGCTACGTGGACGACACCCAG TTCGTCAGATTCGACAGCGACGCCGCCAGCCAGCGGATGGAACCTAGAG CCCCTTGGATCGAGCAGGAAGGCCCCGAGTACTGGGACGGCGAGACACG GAAAGTGAAGGCCCACAGCCAGACCCACAGAGTGGATCTGGGCACCCTG CGGGGCTACTACAATCAGTCTGAGGCCGGCTCCCACACCGTGCAGAGGA TGTACGGCTGTGACGTGGGCAGCGACTGGCGGTTCCTGAGAGGCTACCA CGAGTACGCCTACGACGGCAAGGACTATATCGCCCTGAAAGAGGACCTG CGGAGCTGGACAGCCGCCGATATGGCCGCCCAGACCACCAAGCACAAAT GGGAAGCCGCCCACGTGGCCGAGCAGCTGAGAGCTTATCTGGAAGGCAC CTGTGTGGAATGGCTGCGGAGATACCTGGAAAACGGCAAAGAGACACTG CAGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.6 MMRPIVLVLLFATSALAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQ FVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTL RGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHEYAYDGKDYIALKEDL RSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPG S
Z
Soluble A*24:02-Wt. Nucleotide Sequence (SEQ ID NO. 7) and Amino Acid Sequence (SEQ ID NO. 8)
(132) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-A*24:02 1 domain (underlined below) HLA-A*24:02 2 domain (in bold below) HLA-A*24:02 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(133) TABLE-US-00005 SEQIDNO.7 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGCTCCCACTCCATGAGGTATTTCTCCACATCCGTGTCCCGGCCCGG CCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAG TTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAGGATGGAGCCGCGGG CGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACGAGGAGACAGG GAAAGTGAAGGCCCACTCACAGACTGACCGAGAGAACCTGCGGATCGCG CTCCGCTACTACAACCAGAGCGAGGCCGGTTCTCACACCCTCCAGATGA TGTTTGGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCA CCAGTACGCCTACGACGGCAAGGATTACATCGCCCTGAAAGAGGACCTG CGCTCTTGGACCGCGGCGGACATGGCGGCTCAGATCACCAAGCGCAAGT GGGAGGCGGCCCATGTGGCGGAGCAGCAGAGAGCCTACCTGGAGGGCAC GTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTG CAGCGCACGGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTG ACCATGAGGCCACTCTGAGATGCTGGGCCCTGGGCTTCTACCCTGCGGA GATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CAGCTGTGGTGGTACCTTCTGGAGAGGAGCAGAGATACACCTGCCATGT GCAGCATGAGGGTCTGCCCAAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.8 MMRPIVLVLLFATSALAGSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQ FVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIA LRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDL RSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETL QRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDT ELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPG S
Z
Soluble A*24:02-K.sup.b, Nucleotide Sequence (SEQ ID NO. 9) and Amino Acid Sequence (SEQ ID NO. 10)
(134) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-A*24:02 1 domain (underlined below) HLA-A*24:02 2 domain (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(135) TABLE-US-00006 SEQIDNO.9 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGCTCCCACTCCATGAGGTATTTCTCCACATCCGTGTCCCGGCCCGG CCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAG TTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAGGATGGAGCCGCGGG CGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACGAGGAGACAGG GAAAGTGAAGGCCCACTCACAGACTGACCGAGAGAACCTGCGGATCGCG CTCCGCTACTACAACCAGAGCGAGGCCGGTTCTCACACCCTCCAGATGA TGTTTGGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCA CCAGTACGCCTACGACGGCAAGGATTACATCGCCCTGAAAGAGGACCTG CGCTCTTGGACCGCGGCGGACATGGCGGCTCAGATCACCAAGCGCAAGT GGGAGGCGGCCCATGTGGCGGAGCAGCAGAGAGCCTACCTGGAGGGCAC GTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTG CAGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.10 MMRPIVLVLLFATSALAGSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQ FVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIA LRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDL RSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPG S
Z
Soluble A*24:029115E_K.sup.b Nucleotide Sequence (SEQ ID NO. 11) and Amino Acid Sequence (SEQ ID NO. 12)
(136) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-A*24:02 1 domain (underlined below) HLA-A*24:02 2 domain with Q115E mutation (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(137) TABLE-US-00007 SEQIDNO.11 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGCTCCCACTCCATGAGGTATTTCTCCACATCCGTGTCCCGGCCCGG CCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAG TTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAGGATGGAGCCGCGGG CGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACGAGGAGACAGG GAAAGTGAAGGCCCACTCACAGACTGACCGAGAGAACCTGCGGATCGCG CTCCGCTACTACAACCAGAGCGAGGCCGGTTCTCACACCCTCCAGATGA TGTTTGGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCA CGAGTACGCCTACGACGGCAAGGATTACATCGCCCTGAAAGAGGACCTG CGCTCTTGGACCGCGGCGGACATGGCGGCTCAGATCACCAAGCGCAAGT GGGAGGCGGCCCATGTGGCGGAGCAGCAGAGAGCCTACCTGGAGGGCAC GTGCGTGGACGGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTG CAGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.12 MMRPIVLVLLFATSALAGSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQ FVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIA LRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHEYAYDGKDYIALKEDL RSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADSTLTWQLNGEELSQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPG S
Z
Soluble B*35:019115E-K.sup.b, Nucleotide Sequence (SEQ ID NO. 13) and Amino Acid Sequence (SEQ ID NO. 14)
(138) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-B*35:01 1 domain (underlined below) HLA-B*35:01 2 domain with Q115E mutation (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(139) TABLE-US-00008 SEQIDNO.13 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGCTCCCACTCCATGAGGTATTTCTACACCGCCATGTCCCGGCCCGG CCGCGGGGAGCCCCGCTTCATCGCAGTGGGCTACGTGGACGACACCCAG TTCGTGAGGTTCGACAGCGACGCCGCGAGTCCGAGGACGGAGCCCCGGG CGCCATGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCGGAACACACA GATCTTCAAGACCAACACACAGACTTACCGAGAGAGCCTGCGGAACCTG CGCGGCTACTACAACCAGAGCGAGGCCGGGTCTCACATCATCCAGAGGA TGTATGGCTGCGACCTGGGGCCCGACGGGCGCCTCCTCCGCGGGCATGA CGAGTCCGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTG AGCTCCTGGACCGCGGCGGACACCGCGGCTCAGATCACCCAGCGCAAGT GGGAGGCGGCCCGTGTGGCGGAGCAGCTGAGAGCCTACCTGGAGGGCCT GTGCGTGGAGTGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTG CAGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.14 MMRPIVLVLLFATSALAGSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQ FVRFDSDAASPRTEPRAPWIEQEGPEYWDRNTQIFKTNTQTYRESLRNL RGYYNQSEAGSHIIQRMYGCDLGPDGRLLRGHDESAYDGKDYIALNEDL SSWTAADTAAQITQRKWEAARVAEQLRAYLEGLCVEWLRRYLENGKETL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPG S
Z
Soluble B*40:029115E-K.sup.b. Nucleotide Sequence (SEQ ID NO. 15) and Amino Acid Sequence (SEQ ID NO. 16)
(140) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-B*40:02 1 domain (underlined below) HLA-B*40:02 2 domain with Q115E mutation (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(141) TABLE-US-00009 SEQIDNO.15 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGCAGCCACAGCATGCGGTACTTCCACACCAGCGTGTCCAGACCCGG AAGAGGCGAGCCCAGATTCATCACCGTGGGCTACGTGGACGACACCCTG TTCGTCAGATTCGACAGCGACGCCACCAGCCCCCGGAAAGAACCTAGAG CCCCTTGGATCGAGCAGGAAGGCCCCGAGTACTGGGACAGAGAGACACA GATCAGCAAGACCAACACCCAGACCTACAGAGAGAGCCTGCGGAACCTG CGGGGCTACTACAATCAGAGCGAGGCCGGCTCTCACACCCTGCAGTCTA TGTACGGCTGCGACGTGGGCCCCGATGGCAGACTGCTGAGAGGCCACAA CGAGTACGCCTACGACGGCAAGGACTATATCGCCCTGAACGAGGACCTG CGGAGCTGGACAGCCGCCGATACAGCCGCCCAGATCACCCAGAGAAAGT GGGAGGCCGCCAGAGTGGCCGAACAGCTGAGAGCCTATCTGGAAGGCGA GTGCGTGGAATGGCTGCGGAGATACCTGGAAAACGGCAAAGAGACACTG CAGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.16 MMRPIVLVLLFATSALAGSHSMRYFHTSVSRPGRGEPRFITVGYVDDTL FVRFDSDATSPRKEPRAPWIEQEGPEYWDRETQISKTNTQTYRESLRNL RGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHNEYAYDGKDYIALNEDL RSWTAADTAAQITQRKWEAARVAEQLRAYLEGECVEWLRRYLENGKETL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPG S
Z
Soluble B*44:059115E-K.sup.b, Nucleotide Sequence (SEQ ID NO. 17) and Amino Acid Sequence (SEQ ID NO. 18)
(142) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-B*44:05 1 domain (underlined below) HLA-B*44:05 2 domain with Q115E mutation (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(143) TABLE-US-00010 SEQIDNO.17 ATGATGAGGCCCATCGTGCTGGTGCTGCTGTTCGCCACATCTGCCCTGG CCGGCAGCCACAGCATGCGGTACTTTTACACCGCCATGAGCAGACCCGG CAGAGGCGAGCCCAGATTCATCACCGTGGGCTACGTGGACGACACCCTG TTCGTCAGATTCGACAGCGACGCCACCAGCCCCCGGAAAGAACCTAGAG CCCCTTGGATCGAGCAGGAAGGCCCCGAGTACTGGGACAGAGAGACACA GATCAGCAAGACCAACACCCAGACCTACAGAGAGAACCTGCGGACCGCC CTGCGGTACTACAATCAGTCTGAGGCCGGCTCCCACATCATCCAGCGGA TGTACGGCTGTGACGTGGGCCCCGATGGCAGACTGCTGAGAGGCTACGA CGAGTACGCCTACGACGGCAAGGACTATATCGCCCTGAACGAGGACCTG AGCAGCTGGACAGCCGCCGATACAGCCGCCCAGATCACCCAGAGAAAGT GGGAGGCCGCCAGAGTGGCCGAGCAGGATAGAGCCTATCTGGAAGGCCT GTGCGTGGAAAGCCTGCGGAGATACCTGGAAAACGGCAAAGAGACACTG CAGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.18 MMRPIVLVLLFATSALAGSHSMRYFYTAMSRPGRGEPRFITVGYVDDTL FVRFDSDATSPRKEPRAPWIEQEGPEYWDRETQISKTNTQTYRENLRTA LRYYNQSEAGSHIIQRMYGCDVGPDGRLLRGYDEYAYDGKDYIALNEDL SSWTAADTAAQITQRKWEAARVAEQDRAYLEGLCVESLRRYLENGKETL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADFTLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPG S
Z
Soluble B*07:029115E-K.sup.b, Nucleotide Sequence (SEQ ID NO. 19) and Amino Acid Sequence (SEQ ID NO. 20)
(144) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-B*07:02 1 domain (underlined below) HLA-B*07:02 2 domain with Q115E mutation (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(145) TABLE-US-00011 SEQIDNO.19 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGCAGCCACAGCATGCGGTACTTTTACACCAGCGTGTCCAGACCCGG CAGAGGCGAGCCCAGATTCATCAGCGTGGGCTACGTGGACGACACCCAG TTCGTCAGATTCGACAGCGACGCCGCCAGCCCCAGAGAGGAACCTAGAG CCCCTTGGATCGAGCAGGAAGGCCCCGAGTACTGGGACCGGAACACCCA GATCTACAAGGCCCAGGCCCAGACCGACAGAGAGAGCCTGAGAAACCTG CGGGGCTACTACAACCAGAGCGAGGCCGGCTCTCACACCCTGCAGTCTA TGTACGGCTGCGACGTGGGCCCCGATGGCAGACTGCTGAGAGGCCACGA TGAGTACGCCTACGACGGCAAGGACTATATCGCCCTGAACGAGGACCTG CGGAGCTGGACAGCCGCCGATACAGCCGCCCAGATCACCCAGAGAAAGT GGGAGGCCGCCAGAGAGGCCGAACAGAGAAGGGCCTATCTGGAAGGCGA GTGCGTGGAATGGCTGCGGAGATACCTGGAAAATGGCAAGGACAAGCTG GAACGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.20 MMRPIVLVLLFATSALAGSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQ FVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNL RGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDEYAYDGKDYIALNEDL RSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKL ERTDSPKAHVTHHSRPEDKVTLRCWALGFYPADFFLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLPWEPG S
Z
Soluble B*08:019115E-K.sup.b, Nucleotide Sequence (SEQ ID NO. 21) and Amino Acid Sequence (SEQ ID NO. 22)
(146) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-B*08:01 1 domain (underlined below) HLA-B*08:01 2 domain with Q115E mutation (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(147) TABLE-US-00012 SEQIDNO.21 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGCAGCCACAGCATGCGGTACTTTGACACCGCCATGAGCAGACCCGG CAGAGGCGAGCCCAGATTCATCAGCGTGGGCTACGTGGACGACACCCAG TTCGTCAGATTCGACAGCGACGCCGCCAGCCCCAGAGAGGAACCTAGAG CCCCTTGGATCGAGCAGGAAGGCCCCGAGTACTGGGACCGGAACACCCA GATCTTCAAGACCAATACCCAGACCGACAGAGAGAGCCTGCGGAACCTG CGGGGCTACTACAATCAGAGCGAGGCCGGCTCTCACACCCTGCAGTCTA TGTACGGCTGCGACGTGGGCCCCGATGGCAGACTGCTGAGAGGCCACAA CGAGTACGCCTACGACGGCAAGGACTATATCGCCCTGAACGAGGACCTG CGGAGCTGGACAGCCGCCGATACAGCCGCCCAGATCACCCAGAGAAAGT GGGAGGCCGCCAGAGTGGCCGAGCAGGATAGAGCCTACCTGGAAGGCAC CTGTGTGGAATGGCTGCGGAGATACCTGGAAAATGGCAAGGACACCCTG GAACGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.22 MMRPIVLVLLFATSALAGSHSMRYFDTAMSRPGRGEPRFISVGYVDDTQ FVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIFKTNTQTDRESLRNL RGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHNEYAYDGKDYIALNEDL RSWTAADTAAQITQRKWEAARVAEQDRAYLEGTCVEWLRRYLENGKDTL ERTDSPKAHVTHHSRPEDKVTLRCWALGFYPADFFLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLPWEPG S
Z
Soluble C*05:019115E-K.sup.b, Nucleotide Sequence (SEQ ID NO. 23) and Amino Acid Sequence (SEQ ID NO. 24)
(148) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-C*05:01 1 domain (underlined below) HLA-C*05:01 2 domain with Q115E mutation (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(149) TABLE-US-00013 SEQIDNO.23 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACAAGCGCCCTGG CCTGCTCTCACAGCATGCGCTATTTTTACACGGCAGTTAGTCGGCCTGG GAGGGGTGAGCCGAGATTCATTGCTGTAGGCTACGTAGACGACACTCAA TTTGTACAGTTCGACTCAGACGCTGCTTCACCGCGAGGAGAGCCCAGGG CACCCTGGGTAGAACAAGAAGGGCCCGAATACTGGGATCGAGAAAGCCA GAAGTATAAGAGGCAAGCACAAACTGATCGGGTCAACTTGAGAAAACTG CGAGGCTACTATAATCAAAGTGAGGCAGGATCCCATACACTTCAGAGGA TGTATGGCTGCGACCTTGGTCCAGATGGCCGGCTCCTCAGAGGGTATAA CGAATTTGCATACGACGGGAAGGATTACATAGCTCTCAATGAGGACCTT AGATCATGGACGGCAGCGGATAAGGCAGCCCAAATTACTCAAAGGAAAT GGGAGGCGGCCCGAGAAGCAGAGCAGAGAAGAGCCTACCTGGAAGGTAC ATGCGTGGAGTGGCTTCGGCGCTATCTCGAAAACGGTAAAAAGACATTG CAACGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.24 MMRPIVLVLLFATSALACSHSMRYFYTAVSRPGRGEPRFIAVGYVDDTQ FVQFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVNLRKL RGYYNQSEAGSHTLQRMYGCDLGPDGRLLRGYNEFAYDGKDYIALNEDL RSWTAADKAAQITQRKWEAAREAEQRRAYLEGTCVEWLRRYLENGKKTL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPG S
Z
Soluble C*07:019115E-K.sup.b. Nucleotide Sequence (SEQ ID NO. 25) and Amino Acid Sequence (SEQ ID NO. 26)
(150) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-C*07:01 1 domain (underlined below) HLA-C*07:01 2 domain with Q115E mutation (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(151) TABLE-US-00014 SEQIDNO.25 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACAAGCGCCCTGG CCTGCAGCCACAGCATGCGGTACTTTGACACCGCCGTGTCCAGACCCGG AAGAGGCGAGCCCAGATTCATCAGCGTGGGCTACGTGGACGACACCCAG TTCGTCAGATTCGACAGCGACGCCGCCAGCCCCAGAGGCGAACCTAGAG CACCTTGGGTGGAACAGGAAGGCCCCGAGTACTGGGACAGAGAGACACA GAACTACAAGCGGCAGGCCCAGGCCGACAGAGTGTCCCTGAGAAACCTG CGGGGCTACTACAACCAGAGCGAGGACGGCAGCCACACCCTGCAGAGAA TGTACGGCTGTGACCTGGGCCCCGATGGCAGACTGCTGAGAGGCTACGA TGAGAGCGCCTACGACGGCAAGGACTATATCGCCCTGAACGAGGACCTG CGGAGCTGGACAGCCGCCGATACAGCCGCCCAGATCACCCAGAGAAAAC TGGAAGCCGCCAGAGCCGCCGAGCAGCTGAGAGCTTATCTGGAAGGCAC CTGTGTGGAATGGCTGCGGAGATACCTGGAAAACGGCAAAGAGAGACTG CAGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.26 MMRPIVLVLLFATSALACSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQ FVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNL RGYYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDESAYDGKDYIALNEDL RSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPG S
Z
Soluble C*07:029115E-K.sup.b. Nucleotide Sequence (SEQ ID NO. 27) and Amino Acid Sequence (SEQ ID NO. 28)
(152) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-C*07:02 1 domain (underlined below) HLA-C*07:02 2 domain with Q115E mutation (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(153) TABLE-US-00015 SEQIDNO.27 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCTGCAGCCACAGCATGCGGTACTTTGACACCGCCGTGTCCAGACCCGG AAGAGGCGAGCCCAGATTCATCAGCGTGGGCTACGTGGACGACACCCAG TTCGTCAGATTCGACAGCGACGCCGCCAGCCCCAGAGGCGAACCTAGAG CACCTTGGGTGGAACAGGAAGGCCCCGAGTACTGGGACAGAGAGACACA GAAGTACAAGCGGCAGGCCCAGGCCGACAGAGTGTCCCTGAGAAACCTG CGGGGCTACTACAACCAGAGCGAGGACGGCAGCCACACCCTGCAGAGAA TGAGCGGCTGTGACCTGGGCCCCGATGGCAGACTGCTGAGAGGCTACGA TGAGAGCGCCTACGACGGCAAGGACTATATCGCCCTGAACGAGGACCTG CGGAGCTGGACAGCCGCCGATACAGCCGCCCAGATCACCCAGAGAAAAC TGGAAGCCGCCAGAGCCGCCGAGCAGCTGAGAGCTTATCTGGAAGGCAC CTGTGTGGAATGGCTGCGGAGATACCTGGAAAACGGCAAAGAGACACTG CAGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.28 MMRPIVLVLLFATSALACSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQ FVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVSLRNL RGYYNQSEDGSHTLQRMSGCDLGPDGRLLRGYDESAYDGKDYIALNEDL RSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEFG S
Z
Soluble C*16:019115E-K.sup.b, Nucleotide Sequence (SEQ ID NO. 29) and Amino Acid Sequence (SEQ ID NO. 30)
(154) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-C*16:01 1 domain (underlined below) HLA-C*16:01 2 domain with Q115E mutation (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(155) TABLE-US-00016 SEQIDNO.29 ATGATGAGGCCCATCGTGCTGGTGCTGCTGTTCGCCACATCTGCCCTGG CCTGCAGCCACAGCATGCGGTACTTTTACACCGCCGTGTCCAGACCCGG CAGAGGCGAGCCTAGATTCATTGCCGTGGGCTACGTGGACGACACCCAG TTCGTCAGATTCGACAGCGACGCCGCCAGCCCCAGAGGGGAACCTAGAG CACCTTGGGTGGAACAGGAAGGCCCCGAGTACTGGGACAGAGAGACACA GAAGTACAAGCGGCAGGCCCAGACCGACCGGGTGTCCCTGAGAAACCTG CGGGGCTACTACAACCAGAGCGAGGCCGGCTCTCACACCCTGCAGTGGA TGTACGGCTGCGACCTGGGCCCTGATGGCAGACTGCTGAGAGGCTACGA CGAGTCCGCCTACGACGGCAAGGACTATATCGCCCTGAACGAGGACCTG CGGAGCTGGACAGCCGCCGATACAGCCGCCCAGATCACCCAGAGAAAGT GGGAAGCCGCCAGAGCCGCCGAGCAGCAGAGAGCTTATCTGGAAGGCAC CTGTGTGGAATGGCTGCGGAGATACCTGGAAAACGGCAAAGAGACACTG CAGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.30 MMRPIVLVLLFATSALACSHSMRYFYTAVSRPGRGEPRFIAVGYVDDTQ FVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNL RGYYNQSEAGSHTLQWMYGCDLGPDGRLLRGYDESAYDGKDYIALNEDL RSWTAADTAAQITQRKWEAARAAEQQRAYLEGTCVEWLRRYLENGKETL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPG S
Z
(156) As with the sequences noted above, the present application may similarly be directed to the following sequences:
(157) Soluble B*35:01-Wt, Nucleotide Sequence (SEQ ID NO. 31) and Amino Acid Sequence (SEQ ID NO. 32)
(158) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-B*35:01 1 domain (underlined below) HLA-B*35:01 2 domain (in bold below) HLA-B*35:01 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(159) TABLE-US-00017 SEQIDNO.31 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGCTCCCACTCCATGAGGTATTTCTACACCGCCATGTCCCGGCCCGG CCGCGGGGAGCCCCGCTTCATCGCAGTGGGCTACGTGGACGACACCCAG TTCGTGAGGTTCGACAGCGACGCCGCGAGTCCGAGGACGGAGCCCCGGG CGCCATGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCGGAACACACA GATCTTCAAGACCAACACACAGACTTACCGAGAGAGCCTGCGGAACCTG CGCGGCTACTACAACCAGAGCGAGGCCGGGTCTCACATCATCCAGAGGA TGTATGGCTGCGACCTGGGGCCCGACGGGCGCCTCCTCCGCGGGCATGA CCAGTCCGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTG AGCTCCTGGACCGCGGCGGACACCGCGGCTCAGATCACCCAGCGCAAGT GGGAGGCGGCCCGTGTGGCGGAGCAGCTGAGAGCCTACCTGGAGGGCCT GTGCGTGGAGTGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTG CAGCGCGCGGACCCCCCAAAGACACACGTGACCCACCACCCCGTCTCTG ACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGA GATCACACTGACCTGGCAGCGGGATGGCGAGGACCAAACTCAGGACACT GAGCTTGTGGAGACCAGACCAGCAGGAGATAGAACCTTCCAGAAGTGGG CAGCTGTGGTGGTGCCTTCTGGAGAAGAGCAGAGATACACATGCCATGT ACAGCATGAGGGGCTGCCCAAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.32 MMRPIVLVLLFATSALAGSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQ FVRFDSDAASPRTEPRAPWIEQEGPEYWDRNTQIFKTNTQTYRESLRNL RGYYNQSEAGSHIIQRMYGCDLGPDGRLLRGHDQSAYDGKDYIALNEDL SSWTAADTAAQITQRKWEAARVAEQLRAYLEGLCVEWLRRYLENGKETL QRADPPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDT ELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPG S
Z
Soluble B*35:01-K.sup.b. Nucleotide Sequence (SEQ ID NO. 33) and Amino Acid Sequence (SEQ ID NO. 34)
(160) Sequences are listed in the following order: Signal peptide derived from Fibroin-L (in regular Arial font below) HLA-B*35:01 1 domain (underlined below) HLA-B*35:01 2 domain (in bold below) Mouse K.sup.b 3 domain (in italics below) Flexible GS linker (in bold and underlined below) 6His tag (in bold and italics below)
(161) TABLE-US-00018 SEQIDNO.33 ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGG CCGGCTCCCACTCCATGAGGTATTTCTACACCGCCATGTCCCGGCCCGG CCGCGGGGAGCCCCGCTTCATCGCAGTGGGCTACGTGGACGACACCCAG TTCGTGAGGTTCGACAGCGACGCCGCGAGTCCGAGGACGGAGCCCCGGG CGCCATGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCGGAACACACA GATCTTCAAGACCAACACACAGACTTACCGAGAGAGCCTGCGGAACCTG CGCGGCTACTACAACCAGAGCGAGGCCGGGTCTCACATCATCCAGAGGA TGTATGGCTGCGACCTGGGGCCCGACGGGCGCCTCCTCCGCGGGCATGA CCAGTCCGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTG AGCTCCTGGACCGCGGCGGACACCGCGGCTCAGATCACCCAGCGCAAGT GGGAGGCGGCCCGTGTGGCGGAGCAGCTGAGAGCCTACCTGGAGGGCCT GTGCGTGGAGTGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTG CAGCGCACAGATTCCCCAAAGGCCCATGTGACCCATCACAGCAGACCTG AAGATAAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCTGA CATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGATCCAGGACATG GAGCTTGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGG CATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGTATTACACATGCCATGT GTACCATCAGGGGCTGCCTGAGCCCCTCACCCTGAGATGGGAGCCGGGC AGC TGA SEQIDNO.34 MMRPIVLVLLFATSALAGSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQ FVRFDSDAASPRTEPRAPWIEQEGPEYWDRNTQIFKTNTQTYRESLRNL RGYYNQSEAGSHIIQRMYGCDLGPDGRLLRGHDQSAYDGKDYIALNEDL SSWTAADTAAQITQRKWEAARVAEQLRAYLEGLCVEWLRRYLENGKETL QRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDM ELVETRPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPG S
Z
(162) Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following reference list, are incorporated by reference.
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