METHODS OF IDENTIFYING T CELL RECEPTORS

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name: 4285.009PC02_SL_ST25.txt, Size: 291,794 bytes; and Date of Creation: Jul. 28, 2020) is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure provides methods of identifying MHC class II-specific T cell receptors (“TCRs”).

BACKGROUND OF THE DISCLOSURE

Immunotherapy has emerged as a critical tool in the battle against a variety of diseases, including cancer. T cell therapies are at the forefront of immunotherapeutic development, and adoptive transfer of antitumor T cells has been shown to induce clinical responses in cancer patients. Though many T cell therapies target mutated tumor antigens, the vast majority of neoantigens are not shared and are unique to each patient.

Potential non-mutated antigens outnumber mutated antigens by multiple orders of magnitude. The elucidation of T cell epitopes derived from shared antigens may facilitate the robust development of efficacious and safe adoptive T cell therapies that are readily available to a larger cohort of cancer patients. However, the sheer number of non-mutated antigens and the high polymorphism of HLA genes may have hampered comprehensive analyses of the specificity of antitumor T cell responses toward non-mutated antigens.

SUMMARY OF THE DISCLOSURE

Certain aspects of the present disclosure are directed to a method of identifying an MHC class II-specific T cell receptor (TCR) comprising contacting a T cell with a complex comprising an MHC class II molecule and a peptide; wherein the T cell expresses CD4 and one or more TCRs; wherein the MHC class II molecule comprises an alpha chain and a beta chain, wherein the MHC class II molecule has a higher affinity for CD4 than a naturally occurring MHC class II molecule has for the CD4; and wherein the MHC class II-specific TCR specifically binds the complex comprising the MHC class II molecule and the peptide.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid sequence having one or more mutations relative to a wild-type beta chain of a MHC class II molecule. In some aspects, the alpha chain of the MHC class II molecule comprises an amino acid sequence having one or more mutations relative to a wild-type alpha chain of a MHC class II molecule. In some aspects, the one or more mutations comprise a substitution mutation.

In some aspects, the MHC class II molecule is an HLA-DP, HLA-DQ, or HLA-DR allele, or any combination thereof. In some aspects, (i) the beta chain of the HLA class II molecule is an HLA-DP allele, (ii) the alpha chain of the HLA class II molecule is an HLA-DP allele, or (iii) both (i) and (ii). In some aspects, the beta chain of the HLA class II molecule is a DP1, DP2, DP3, DP4, DP5, DP6, DP8, or DP9 allele.

In some aspects, the beta chain of the MHC class II molecule comprises an HLA allele selected from the group consisting of DPB1*01, DPB1*02, DPB1*03, DPB1*04, DPB1*05, DPB1*06, DPB1*08, DPB1*09, DPB1*10, DPB1*100, DPB1*101, DPB1*102, DPB1*103, DPB1*104, DPB1*105, DPB1*106, DPB1*107, DPB1*108, DPB1*109, DPB1*110, DPB1*111, DPB1*112, DPB1*113, DPB1*114, DPB1*115, DPB1*116, DPB1*117, DPB1*118, DPB1*119, DPB1*11, DPB1*120, DPB1*121, DPB1*122, DPB1*123, DPB1*124, DPB1*125, DPB1*126, DPB1*127, DPB1*128, DPB1*129, DPB1*130, DPB1*131, DPB1*132, DPB1*133, DPB1*134, DPB1*135, DPB1*136, DPB1*137, DPB1*138, DPB1*139, DPB1*13, DPB1*140, DPB1*141, DPB1*142, DPB1*143, DPB1*144, DPB1*145, DPB1*146, DPB1*147, DPB1*148, DPB1*149, DPB1*14, DPB1*150, DPB1*151, DPB1*152, DPB1*153, DPB1*154, DPB1*155, DPB1*156, DPB1*157, DPB1*158, DPB1*159, DPB1*15, DPB1*160, DPB1*161, DPB1*162, DPB1*163, DPB1*164, DPB1*165, DPB1*166, DPB1*167, DPB1*168, DPB1*169, DPB1*16, DPB1*170, DPB1*171, DPB1*172, DPB1*173, DPB1*174, DPB1*175, DPB1*176, DPB1*177, DPB1*178, DPB1*179, DPB1*17, DPB1*180, DPB1*181, DPB1*182, DPB1*183, DPB1*184, DPB1*185, DPB1*186, DPB1*187, DPB1*188, DPB1*189, DPB1*18, DPB1*190, DPB1*191, DPB1*192, DPB1*193, DPB1*194, DPB1*195, DPB1*196, DPB1*197, DPB1*198, DPB1*199, DPB1*19, DPB1*200, DPB1*201, DPB1*202, DPB1*203, DPB1*204, DPB1*205, DPB1*206, DPB1*207, DPB1*208, DPB1*209, DPB1*20, DPB1*210, DPB1*211, DPB1*212, DPB1*213, DPB1*214, DPB1*215, DPB1*216, DPB1*217, DPB1*218, DPB1*219, DPB1*21, DPB1*220, DPB1*221, DPB1*222, DPB1*223, DPB1*224, DPB1*225, DPB1*226, DPB1*227, DPB1*228, DPB1*229, DPB1*22, DPB1*230, DPB1*231, DPB1*232, DPB1*233, DPB1*234, DPB1*235, DPB1*236, DPB1*237, DPB1*238, DPB1*239, DPB1*23, DPB1*240, DPB1*241, DPB1*242, DPB1*243, DPB1*244, DPB1*245, DPB1*246, DPB1*247, DPB1*248, DPB1*249, DPB1*24, DPB1*250, DPB1*251, DPB1*252, DPB1*253, DPB1*254, DPB1*255, DPB1*256, DPB1*257, DPB1*258, DPB1*259, DPB1*25, DPB1*260, DPB1*261, DPB1*262, DPB1*263, DPB1*264, DPB1*265, DPB1*266, DPB1*267, DPB1*268, DPB1*269, DPB1*26, DPB1*270, DPB1*271, DPB1*272, DPB1*273, DPB1*274, DPB1*275, DPB1*276, DPB1*277, DPB1*278, DPB1*279, DPB1*27, DPB1*280, DPB1*281, DPB1*282, DPB1*283, DPB1*284, DPB1*285, DPB1*286, DPB1*287, DPB1*288, DPB1*289, DPB1*28, DPB1*290, DPB1*291, DPB1*292, DPB1*293, DPB1*294, DPB1*295, DPB1*296, DPB1*297, DPB1*298, DPB1*299, DPB1*29, DPB1*300, DPB1*301, DPB1*302, DPB1*303, DPB1*304, DPB1*305, DPB1*306, DPB1*307, DPB1*308, DPB1*309, DPB1*30, DPB1*310, DPB1*311, DPB1*312, DPB1*313, DPB1*314, DPB1*315, DPB1*316, DPB1*317, DPB1*318, DPB1*319, DPB1*31, DPB1*320, DPB1*321, DPB1*322, DPB1*323, DPB1*324, DPB1*325, DPB1*326, DPB1*327, DPB1*328, DPB1*329, DPB1*32, DPB1*330, DPB1*331, DPB1*332, DPB1*333, DPB1*334, DPB1*335, DPB1*336, DPB1*337, DPB1*338, DPB1*339, DPB1*33, DPB1*340, DPB1*341, DPB1*342, DPB1*343, DPB1*344, DPB1*345, DPB1*346, DPB1*347, DPB1*348, DPB1*349, DPB1*34, DPB1*350, DPB1*351, DPB1*352, DPB1*353, DPB1*354, DPB1*355, DPB1*356, DPB1*357, DPB1*358, DPB1*359, DPB1*35, DPB1*360, DPB1*361, DPB1*362, DPB1*363, DPB1*364, DPB1*365, DPB1*366, DPB1*367, DPB1*368, DPB1*369, DPB1*36, DPB1*370, DPB1*371, DPB1*372, DPB1*373, DPB1*374, DPB1*375, DPB1*376, DPB1*377, DPB1*378, DPB1*379, DPB1*37, DPB1*380, DPB1*381, DPB1*382, DPB1*383, DPB1*384, DPB1*385, DPB1*386, DPB1*387, DPB1*388, DPB1*389, DPB1*38, DPB1*390, DPB1*391, DPB1*392, DPB1*393, DPB1*394, DPB1*395, DPB1*396, DPB1*397, DPB1*398, DPB1*399, DPB1*39, DPB1*400, DPB1*401, DPB1*402, DPB1*403, DPB1*404, DPB1*405, DPB1*406, DPB1*407, DPB1*408, DPB1*409, DPB1*40, DPB1*410, DPB1*411, DPB1*412, DPB1*413, DPB1*414, DPB1*415, DPB1*416, DPB1*417, DPB1*418, DPB1*419, DPB1*41, DPB1*420, DPB1*421, DPB1*422, DPB1*423, DPB1*424, DPB1*425, DPB1*426, DPB1*427, DPB1*428, DPB1*429, DPB1*430, DPB1*431, DPB1*432, DPB1*433, DPB1*434, DPB1*435, DPB1*436, DPB1*437, DPB1*438, DPB1*439, DPB1*440, DPB1*441, DPB1*442, DPB1*443, DPB1*444, DPB1*445, DPB1*446, DPB1*447, DPB1*448, DPB1*449, DPB1*44, DPB1*450, DPB1*451, DPB1*452, DPB1*453, DPB1*454, DPB1*455, DPB1*456, DPB1*457, DPB1*458, DPB1*459, DPB1*45, DPB1*460, DPB1*461, DPB1*462, DPB1*463, DPB1*464, DPB1*465, DPB1*466, DPB1*467, DPB1*468, DPB1*469, DPB1*46, DPB1*470, DPB1*471, DPB1*472, DPB1*473, DPB1*474, DPB1*475, DPB1*476, DPB1*477, DPB1*478, DPB1*479, DPB1*47, DPB1*480, DPB1*481, DPB1*482, DPB1*483, DPB1*484, DPB1*485, DPB1*486, DPB1*487, DPB1*488, DPB1*489, DPB1*48, DPB1*490, DPB1*491, DPB1*492, DPB1*493, DPB1*494, DPB1*495, DPB1*496, DPB1*497, DPB1*498, DPB1*499, DPB1*49, DPB1*500, DPB1*501, DPB1*502, DPB1*503, DPB1*504, DPB1*505, DPB1*506, DPB1*507, DPB1*508, DPB1*509, DPB1*50, DPB1*510, DPB1*511, DPB1*512, DPB1*513, DPB1*514, DPB1*515, DPB1*516, DPB1*517, DPB1*518, DPB1*519, DPB1*51, DPB1*520, DPB1*521, DPB1*522, DPB1*523, DPB1*524, DPB1*525, DPB1*526, DPB1*527, DPB1*528, DPB1*529, DPB1*52, DPB1*530, DPB1*531, DPB1*532, DPB1*533, DPB1*534, DPB1*535, DPB1*536, DPB1*537, DPB1*538, DPB1*539, DPB1*53, DPB1*540, DPB1*541, DPB1*542, DPB1*543, DPB1*544, DPB1*545, DPB1*546, DPB1*547, DPB1*548, DPB1*549, DPB1*54, DPB1*550, DPB1*551, DPB1*552, DPB1*553, DPB1*554, DPB1*555, DPB1*556, DPB1*557, DPB1*558, DPB1*559, DPB1*55, DPB1*560, DPB1*561, DPB1*562, DPB1*563, DPB1*564, DPB1*565, DPB1*566, DPB1*567, DPB1*568, DPB1*569, DPB1*56, DPB1*570, DPB1*571, DPB1*572, DPB1*573, DPB1*574, DPB1*575, DPB1*576, DPB1*577, DPB1*578, DPB1*579, DPB1*57, DPB1*580, DPB1*581, DPB1*582, DPB1*583, DPB1*584, DPB1*585, DPB1*586, DPB1*587, DPB1*588, DPB1*589, DPB1*58, DPB1*590, DPB1*591, DPB1*592, DPB1*593, DPB1*594, DPB1*595, DPB1*596, DPB1*597, DPB1*598, DPB1*599, DPB1*59, DPB1*600, DPB1*601, DPB1*602, DPB1*603, DPB1*604, DPB1*605, DPB1*606, DPB1*607, DPB1*608, DPB1*609, DPB1*60, DPB1*610, DPB1*611, DPB1*612, DPB1*613, DPB1*614, DPB1*615, DPB1*616, DPB1*617, DPB1*618, DPB1*619, DPB1*61, DPB1*620, DPB1*621, DPB1*622, DPB1*623, DPB1*624, DPB1*625, DPB1*626, DPB1*627, DPB1*628, DPB1*629, DPB1*62, DPB1*630, DPB1*631, DPB1*632, DPB1*633, DPB1*634, DPB1*635, DPB1*636, DPB1*637, DPB1*638, DPB1*639, DPB1*63, DPB1*640, DPB1*641, DPB1*642, DPB1*643, DPB1*644, DPB1*645, DPB1*646, DPB1*647, DPB1*648, DPB1*649, DPB1*64, DPB1*650, DPB1*651, DPB1*652, DPB1*653, DPB1*654, DPB1*655, DPB1*656, DPB1*657, DPB1*658, DPB1*659, DPB1*65, DPB1*660, DPB1*661, DPB1*662, DPB1*663, DPB1*664, DPB1*665, DPB1*666, DPB1*667, DPB1*668, DPB1*669, DPB1*66, DPB1*670, DPB1*671, DPB1*672, DPB1*673, DPB1*674, DPB1*675, DPB1*676, DPB1*677, DPB1*678, DPB1*679, DPB1*67, DPB1*680, DPB1*681, DPB1*682, DPB1*683, DPB1*684, DPB1*685, DPB1*686, DPB1*687, DPB1*688, DPB1*689, DPB1*68, DPB1*690, DPB1*691, DPB1*692, DPB1*693, DPB1*694, DPB1*695, DPB1*696, DPB1*697, DPB1*698, DPB1*699, DPB1*69, DPB1*700, DPB1*701, DPB1*702, DPB1*703, DPB1*704, DPB1*705, DPB1*706, DPB1*707, DPB1*708, DPB1*709, DPB1*70, DPB1*710, DPB1*711, DPB1*712, DPB1*713, DPB1*714, DPB1*715, DPB1*716, DPB1*717, DPB1*718, DPB1*719, DPB1*71, DPB1*720, DPB1*721, DPB1*722, DPB1*723, DPB1*724, DPB1*725, DPB1*726, DPB1*727, DPB1*728, DPB1*729, DPB1*72, DPB1*730, DPB1*731, DPB1*732, DPB1*733, DPB1*734, DPB1*735, DPB1*736, DPB1*737, DPB1*738, DPB1*739, DPB1*73, DPB1*740, DPB1*741, DPB1*742, DPB1*743, DPB1*744, DPB1*745, DPB1*746, DPB1*747, DPB1*748, DPB1*749, DPB1*74, DPB1*750, DPB1*751, DPB1*752, DPB1*753, DPB1*754, DPB1*755, DPB1*756, DPB1*757, DPB1*758, DPB1*759, DPB1*75, DPB1*760, DPB1*761, DPB1*762, DPB1*763, DPB1*764, DPB1*765, DPB1*766, DPB1*767, DPB1*768, DPB1*769, DPB1*76, DPB1*770, DPB1*771, DPB1*772, DPB1*773, DPB1*774, DPB1*775, DPB1*776, DPB1*777, DPB1*778, DPB1*779, DPB1*77, DPB1*780, DPB1*781, DPB1*782, DPB1*783, DPB1*784, DPB1*785, DPB1*786, DPB1*787, DPB1*788, DPB1*789, DPB1*78, DPB1*790, DPB1*791, DPB1*792, DPB1*794, DPB1*795, DPB1*796, DPB1*797, DPB1*798, DPB1*799, DPB1*79, DPB1*800, DPB1*801, DPB1*802, DPB1*803, DPB1*804, DPB1*805, DPB1*806, DPB1*807, DPB1*808, DPB1*809, DPB1*80, DPB1*810, DPB1*811, DPB1*812, DPB1*813, DPB1*814, DPB1*815, DPB1*816, DPB1*817, DPB1*818, DPB1*819, DPB1*81, DPB1*820, DPB1*821, DPB1*822, DPB1*823, DPB1*824, DPB1*825, DPB1*826, DPB1*827, DPB1*828, DPB1*829, DPB1*82, DPB1*830, DPB1*831, DPB1*832, DPB1*833, DPB1*834, DPB1*835, DPB1*836, DPB1*837, DPB1*838, DPB1*839, DPB1*83, DPB1*840, DPB1*841, DPB1*842, DPB1*843, DPB1*844, DPB1*845, DPB1*846, DPB1*847, DPB1*848, DPB1*849, DPB1*84, DPB1*850, DPB1*851, DPB1*852, DPB1*853, DPB1*854, DPB1*855, DPB1*856, DPB1*857, DPB1*858, DPB1*859, DPB1*85, DPB1*860, DPB1*861, DPB1*862, DPB1*863, DPB1*864, DPB1*865, DPB1*866, DPB1*867, DPB1*868, DPB1*869, DPB1*86, DPB1*870, DPB1*871, DPB1*872, DPB1*873, DPB1*874, DPB1*875, DPB1*876, DPB1*877, DPB1*878, DPB1*879, DPB1*87, DPB1*880, DPB1*881, DPB1*882, DPB1*883, DPB1*884, DPB1*885, DPB1*886, DPB1*887, DPB1*888, DPB1*889, DPB1*88, DPB1*890, DPB1*891, DPB1*892, DPB1*893, DPB1*894, DPB1*895, DPB1*896, DPB1*897, DPB1*898, DPB1*899, DPB1*89, DPB1*900, DPB1*901, DPB1*902, DPB1*903, DPB1*904, DPB1*905, DPB1*906, DPB1*907, DPB1*908, DPB1*909, DPB1*90, DPB1*910, DPB1*911, DPB1*912, DPB1*913, DPB1*914, DPB1*915, DPB1*916, DPB1*917, DPB1*918, DPB1*919, DPB1*91, DPB1*920, DPB1*921, DPB1*922, DPB1*923, DPB1*924, DPB1*925, DPB1*926, DPB1*927, DPB1*928, DPB1*929, DPB1*92, DPB1*930, DPB1*931, DPB1*932, DPB1*933, DPB1*934, DPB1*935, DPB1*936, DPB1*937, DPB1*938, DPB1*939, DPB1*93, DPB1*940, DPB1*941, DPB1*942, DPB1*943, DPB1*944, DPB1*945, DPB1*946, DPB1*947, DPB1*948, DPB1*949, DPB1*94, DPB1*950, DPB1*951, DPB1*952, DPB1*953, DPB1*954, DPB1*955, DPB1*956, DPB1*957, DPB1*958, DPB1*959, DPB1*95, DPB1*960, DPB1*961, DPB1*962, DPB1*963, DPB1*964, DPB1*965, DPB1*96, DPB1*97, DPB1*98, and DPB1*99 allele.

In some aspects, the alpha chain of the MHC class II molecule comprises an HLA-DPA1*01, HLA-DPA1*02, HLA-DPA1*03, or HLA-DPA1*04 allele.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1. In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1.

Certain aspects of the present disclosure are directed to a method of identifying a MHC class II-specific T cell receptor (TCR) comprising contacting a T cell with a complex comprising an MHC class II molecule and a peptide; wherein the T cell expresses CD4 and one or more TCRs; wherein the MHC class II molecule comprises an alpha chain and a beta chain, wherein the beta chain of the MHC class II molecule comprises (i) an amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, (ii) an amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, or (iii) both (i) and (ii); and wherein the MHC class II-specific TCR specifically binds the complex comprising the MHC class II molecule and the peptide.

In some aspects, the MHC class II molecule has a higher affinity for CD4 than a naturally occurring MHC class II molecule has for CD4.

In some aspects, the amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1 comprises a hydrophobic side chain. In some aspects, the amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1 is selected from the group consisting of an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In some aspects, the amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1 is a tryptophan.

In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1 comprises a hydrophobic side chain. In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1 is selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1 is a methionine.

In some aspects, (i) the beta chain of the HLA class II molecule is an HLA-DQ allele, (ii) the alpha chain of the HLA class II molecule is an HLA-DQ allele, or (iii) both (i) and (ii). In some aspects, the beta chain of the HLA class II molecule comprises a DQ2, DQ3, DQ4, DQ5, or DQ6 allele. In some aspects, the beta chain of the MHC class II molecule comprises an HLA-DQB1*02, HLA-DQB1*03, HLA-DQB1*04, HLA-DQB1*05, or HLA-DQB1*06 allele. In some aspects, the alpha chain of the MHC class II molecule comprises an HLA-DQA1*01, HLA-DQA1*02, HLA-DQA1*03, HLA-DQA1*04, HLA-DQA1*05, or HLA-DQA1*06 allele.

In some aspects, the beta chain of the MHC class II molecule comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 11; (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11; and (c) at least three of: (i) an amino acid other than asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11, (ii) an amino acid other than isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11, (iii) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11, and (iv) an amino acid other than proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11.

In some aspects, the beta chain of the MHC class II molecule comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 11; (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11; (c) an amino acid other than asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11; (d) an amino acid other than isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11; (e) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11; and (f) an amino acid other than proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11.

In some aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 11 comprises a hydrophobic side chain. In some aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 11 is selected from the group consisting of an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In some aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 11 is a tryptophan.

In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11 comprises a hydrophobic side chain. In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11 is selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11 is a methionine.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11. In some aspects, the amino acid other than asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11 is selected from a serine, a threonine, and a glutamine. In some aspects, the amino acid other than asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11 is a glutamine.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11. In some aspects, the amino acid other than isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11 is selected from an alanine, a valine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In some aspects, the amino acid other than isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11 is valine.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11. In some aspects, the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11 is selected from an arginine, a histidine, and a lysine. In some aspects, the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11 is a histidine.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11. In some aspects, the amino acid other than proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11 is selected from a serine, a threonine, an asparagine, and a glutamine. In some aspects, the amino acid other than proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11 is a glutamine.

In some aspects, (i) the beta chain of the HLA class II molecule is an HLA-DR allele, (ii) the alpha chain of the HLA class II molecule is an HLA-DR allele, of (iii) both (i) and (ii).

In some aspects, the beta chain of the HLA class II molecule comprises a DR2, DR3, DR4, DR5, DR6, DR7, DR8, DR9, DR10, DR11, DR12, DR13, DR14, DR15, or DR16 allele. In some aspects, the beta chain of the MHC class II molecule comprises an HLA allele selected from the group consisting of DRB1*01, DRB1*03, DRB1*04, DRB1*07, DRB1*08, DRB1*09, DRB1*10, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*15, and DRB1*16. In some aspects, the alpha chain of the MHC class II molecule comprises an HLA-DRA1*01 allele.

In some aspects, the beta chain comprises: (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19; (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19; and (c) at least two of: (i) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19, (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19.

In some aspects, the beta chain comprises: (c) at least three of: (i) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19, (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19.

In some aspects, the beta chain comprises: (c) at least four of: (i) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19, (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19.

In some aspects, the beta chain comprises: (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19, (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19, (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, (d) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (e) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (f) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19, (g) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, and (h) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19.

In some aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19 comprises a hydrophobic side chain. In some aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19 is selected from the group consisting of an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In some aspects, the amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19 is a tryptophan.

In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19 comprises a hydrophobic side chain. In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19 is selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19 is a methionine.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19. In some aspects, the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19 is selected from an arginine, a histidine, and a lysine. In some aspects, the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19 is a histidine.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19. In some aspects, the amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19 is selected from a serine, a threonine, and a glutamine. In some aspects, the amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19 is a threonine.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19. In some aspects, the amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19 is selected from a serine, an asparagine, a threonine, and a glutamine. In some aspects, the amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19 is a glutamine.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19. In some aspects, the amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19 is selected from an alanine, a valine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In some aspects, the amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19 is an isoleucine.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19. In some aspects, the amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19 is selected from an alanine, a valine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In some aspects, the amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19 is a methionine.

In some aspects, the beta chain of the MHC class II molecule comprises an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19. In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19 is selected from a serine, an asparagine, a threonine, and a glutamine. In some aspects, the amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19 is a threonine.

In some aspects, the beta chain comprises: (a) a tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 19, (b) a methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19, (c) a histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, and (d) an isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19.

In some aspects, the naturally occurring MHC class II molecule comprises: (a) a leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1 or amino acid residue 114 of SEQ ID NO: 11 or 19, (b) a valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1 or amino acid residue 143 of SEQ ID NO: 11 or 19, (c) an asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11; (d) an isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11; (e) a serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11 or 19; and (f) a proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11 (g) a lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (h) a glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (i) a threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19, (j) a threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, (k) a valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19, or (1) any combination of (a) to (k).

In some aspects, the MHC class II molecule is a dimer. In some aspects, the MHC class II molecule is a trimer. In some aspects, the MHC class II molecule is a tetramer. In some aspects, the peptide comprises a fragment of a protein. In some aspects, the protein is expressed by a diseased cell. In some aspects, the protein is expressed by a tumor cell.

In some aspects, the peptide comprises at least about 10 amino acids. In some aspects, the peptide comprises about 10 to about 100 amino acids, about 10 to about 90 amino acids, about 10 to about 80 amino acids, about 10 to about 70 amino acids, about 10 to about 60 amino acids, about 10 to about 50 amino acids, about 10 to about 40 amino acids, about 10 to about 30 amino acids, about 10 to about 25 amino acids, about 10 to about 20 amino acids, about 10 to about 15 amino acids, about 15 to about 100 amino acids, 20 to about 100 amino acids, 25 to about 100 amino acids, 30 to about 100 amino acids, 35 to about 100 amino acids, 40 to about 100 amino acids, 50 to about 100 amino acids, 60 to about 100 amino acids, 70 to about 100 amino acids, 80 to about 100 amino acids, or 90 to about 100 amino acids.

In some aspects, the peptide comprises about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids, about 50 amino acids, about 55 amino acids, about 60 amino acids, about 65 amino acids, about 70 amino acids, about 75 amino acids, about 80 amino acids, about 85 amino acids, about 90 amino acids, about 95 amino acids, or about 100 amino acids.

In some aspects, the MHC class II molecule is expressed on the surface of an antigen presenting cell.

In some aspects, the T cell is obtained from a human subject. In some aspects, the T cell is a tumor infiltrating lymphocyte (TIL).

In some aspects, the MHC class II molecule has an affinity for CD4 that is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 100-fold higher than the binding affinity of a naturally occurring MHC class II molecule to CD4.

In some aspects, the method further comprises selecting the T cell that is bound by the MHC class II molecule. In some aspects, the method further comprises isolating the TCR that is bound to the MHC class II molecule. In some aspects, the method further comprises sequencing the TCR. In some aspects, the method further comprises cloning the TCR. In some aspects, the method further comprises recombinantly expressing the TCR in a host cell.

In some aspects, the MHC class II molecule binds CD4 with a K_Dof less than about 100 μM, less than about 50 μM, less than about 20 μM, or less than about 10 μM. In some aspects, the MHC class II molecule binds CD4 with a K_Dof about 14 μM or less. In some aspects, the MHC class II molecule binds CD4 with a K_Dof about 8.9 μM or less.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1V are graphical representations of data illustrating that affinity-matured DP4^L112W/V141Mmolecules exhibit an enhanced CD4 binding ability. FIGS. 1A-1F are histograms showing the results of HLA class II-null K562 cells stably expressing the wild-type DPα chain (DPA1*01:03) transduced with blank, wild-type, or mutant DPβ chain (DPB1*04:01) harboring L112W, V114M, V141M, and M158I substitutions (DP4^{L112W/V114M/V141M/M158I}) and stained with an anti-class II mAb and soluble CD4 (sCD4). FIG. 1G is a bar graph summarizing the binding affinity for sCD4 (MFI; y-axis) of all possible DP4 reversion mutants, which were similarly expressed and stained with sCD4 as FIGS. 1A-1F. FIG. 1H shows the affinity between DP4^L112W/V141Mand CD4 as quantified by steady state analysis. FIG. 1I shows the results of an IL-2 EPISPOT assay of DP4/WT1 TCR, clone 9-transduced Jurkat 76 and Jurkat 76/CD4 cells stimulated by wild-type DP4 or DP4^L112W/V141M-expressing aAPCs pulsed with graded concentrations of the DP4/WT1 peptide. FIGS. 1J-1W are histograms representing staining of K562 cells expressing DP^L112W/V141Malleles (as indicated) with an anti-class II mAb and sCD4. Open histograms represent the isotype control staining. *P<0.05 by Student's t-test. Bars and error bars represent the mean±SD of results in triplicate experiments. At least 2 independent experiments were performed. FIGS. 1X-IAA are histograms showing wild-type DP4 and DP4L112W/V141M molecules on the surface of K562 cells that were detected with the indicated anti-HLA class II antibodies. Staining of control cells devoid of Class II expression is shown in solid gray. FIGS. 1AB-1BH are histograms showing aAPCs expressing the indicated DP4 or class II parental cells that were stained with sCD4 at the indicated concentrations. FIG. 1BI shows the quantification of aAPCs expressing wild-type DP4 or DP4^L112W/V141Mat the indicated concentrations. Error bars represent the mean±standard deviation of experiments performed in triplicate. FIG. 1BJ is a biolayer interferometry sensogram showing the interaction of biotinylated wild-type DP4 (ligand) with sCD4 (analyte) over a range of concentrations. FIG. 1BK is a biolayer interferometry sensogram showing the interaction of biotinylated DP4^L112W/V141M(ligand) with sCD4 (analyte) over a range of concentrations. Experiments in FIGS. 1BJ and 1BI were performed in parallel. All data are representative of two independent experiments.

FIGS. 2A-2D are ribbon diagrams of a model structure of DP4^L112W/V141Mand the human CD4 complex. FIGS. 2A-2B are two orientations of the ternary complex model structure of DPA1*01:03, DPB1*04:01, and CD4, as indicated. The DPB1*04:01-CD4 binding interface is enclosed in a dashed square (FIG. 2B). FIGS. 2C-2D provide close-up views of the CD4 binding interface of wild-type DP4 (FIG. 2C) and DP4^L112W/V141M(FIG. 2D). The side chains of interacting residues are shown as ball-and-stick representations (FIGS. 2C-2D).

FIGS. 3A-3P are graphical representations of data illustrating that DP4^L112W/V141Mdimers stain cognate TCRs expressed in human primary CD4⁺T cells. Primary T cells were transduced with either DP4/MAGE-A3_243-258(R12C9; FIGS. 3E-3H), DP4/WT1_328-348(clone 9; FIGS. 3I-3L), or DP4/NY-ESO-1_157-170(5B8; FIGS. 3M-3P) TCR and stained with the indicated DP4^L112W/V141Mdimers (FIGS. 3B-3D, 3F-3H, 3J-3L, and 3N-3P).

FIGS. 4A-4D are scatter plots illustrating costaining of R12C9-transduced CD4⁺T cells stained with DP4^L112W/V141Mdimer and an anti-V022 mAb. Note that R12C9 expresses V022. FIGS. 4E-4H are scatter plots illustrating costaining of Clone 9-transduced CD4⁺T cells double-stained with DP4^L112W/V141Mdimer and an anti-NGFR mAb. Note that clone 9 and ΔNGFR genes are fused with P2A.

FIGS. 5A-5P are scatter plots illustrating costaining of Clone 9- (FIGS. 5A-5H) and 5B8- (FIGS. 5I-5P) transduced primary T cells stained with 5 μg/ml conventional wild-type DP4 tetramers and DP4^L112W/V141Mdimers. At least 2 independent experiments were performed.

FIGS. 6A-6F are bar graphs illustrating the results of comprehensive screening with DP4^L112W/V141Mdimers, which identified an array of novel DP4-restricted tumor-associated antigens. Peripheral CD4⁺T cells were purified from six DP4⁺ melanoma patients and stimulated with DP4-expressing aAPCs individually pulsed with 196 distinct peptides derived from tumor-associated antigens and stained with cognate DP4^L112W/V141Mdimers. The results using the 30 peptides with the highest positivity values are shown in FIGS. 6A-6B. The results for the remaining 166 peptides are shown in FIGS. 6C-6F. Each gating was set so that control dimer staining showed <0.2% positivity. Positive dimer staining was defined as staining exceeding the control dimer staining by 3 standard deviations, as shown by the dashed line (>0.6%).

FIGS. 7A-7L are graphical representations of DP4^L112W/V141Mdimer staining of peptide-specific CD4⁺ T cells from melanoma patients. Primary CD4⁺ T cells were purified from six DP4⁺ melanoma patients and stimulated with DP4-expressing aAPCs individually pulsed with 196 distinct peptides derived from tumor-associated antigens and stained with cognate DP4^L112W/V141Mdimers as shown in FIGS. 6A-6F. Examples of DP4^L112W/V141Mdimer staining are shown. *P<0.05 by Student's t-test. n.s., not significant. At least 2 independent experiments were performed.

FIGS. 8A-8X are graphical representations of data illustrating that DP4-restricted TCRs isolated from DP4^L112W/V141Mdimer-positive cells and reconstituted in human TCR-defective CD4⁺ T cells were functional in a DP4-restricted and antigen-specific manner. 03-CCND1_219-238(FIGS. 8A-8D), 05-HSD17B12_225-244and 09-HSD17B12_225-244(FIGS. 8E-8J), 05-LGSN_296-315(FIGS. 8K-8N), 03-MAGE-A2_108-127and 06-MAGE-A2_108-127(FIGS. 80-8T), and 05-MUC5AC_4922-4941(FIGS. 8U-8X) were cloned from DP4^L112W/V141Mdimer-positive cells, reconstituted in TCR-defective Jurkat 76/CD4 cells, and stained by the respective DP4^L112W/V141Mdimers.

FIGS. 9A-9G are bar graphs illustrating the results of IL-2 EPISPOT assays of 03-CCND1_219-238(FIG. 9A), 05-HSD17B12_225-244(FIG. 9B), 09-HSD17B12_225-244(FIG. 9C), 05-LGSN_296-315(FIG. 9D), 03-MAGE-A2_108-127(FIG. 9E), 06-MAGE-A2_108-127(FIG. 9F), and 05-MUC5AC_4922-4941(FIG. 9G) were stimulated by aAPCs pulsed with the respective peptides in IL-2 ELISPOT assays. DP4/WT1 (clone 9) TCR was used as a negative control. At least 2 independent experiments were performed. *, P<0.05 by Student's t-test. Bars and error bars represent the mean±SD of results in triplicate experiments.

FIGS. 10A-10Q are graphical representations of data showing that DP4-restricted TCRs isolated from DP4^L112W/V141Mdimer-positive cells and reconstituted in human primary CD4⁺ T cells were functional in a DP4-restricted and antigen-specific manner. 03-CCND1_219-238(FIGS. 10A-10D and 10O), 03-MAGE-A2_108-127and 06-MAGE-A2_108-127(FIGS. 10E-10J and 10P) and 05-MUC5AC_4922-4941(FIGS. 10K-10N and 10Q) were retrovirally transduced into human primary CD4⁺ T cells and stained with the respective DP4^L112W/V141Mdimers (FIGS. 10A-10N). *P<0.05 by Student's t-test. n.s., not significant. At least 2 independent experiments were performed. *, P<0.05 by Student's t-test. Bars and error bars represent the mean±SD of results in triplicate experiments.

FIGS. 11A-11E present data showing that DP4-restricted TCRs cloned from melanoma patients recognized peptides endogenously processed and presented by K562-based aAPCs. FIGS. 11A-11B are images of gel chromatography showing CCDN1 (FIG. 11A) and MAGE-A2 (FIG. 11B) endogenously expressed in K562-derived aAPC cells. FIGS. 11C-11D are bar graphs showing the results of IFN-7 ELISPOT assays of human primary T cells retrovirally transduced with 03-CCND1_219-238(FIG. 11C) or 06-MAGE-A2_108-127(FIG. 11D) and stimulated with peptide-unpulsed HLA-null or DP4-aAPCs (FIGS. 11C-11D). FIG. 11E is a bar graph showing the results of an IFN-7 ELISPOT assay of human primary T cells retrovirally transduced with 05-MUC5AC_4922-4941TCR and stimulated with MUC5AC_4914-4949minigene-transduced and peptide-unpulsed HLA-null or DP4-aAPCs. At least 2 independent experiments were performed. *, P<0.05 by Student's t-test. Bars and error bars represent the mean±SD of results in triplicate experiments.

FIGS. 12A-12E present data showing that 06-MAGE-A2_108-127TCR recognizes melanoma cell lines in a DP4- and MAGE-A2-dependent manner. FIG. 12A is an image of western blot showing endogenous MAGE-A2 expression in K562 cells and the indicated melanoma cell lines. FIGS. 12B-12E are bar graphs showing data from IFN-7 ELISPOT assays of primary human T cells transduced with 06-MAGE-A2_108-127TCR stimulated with SK-MEL-21 (DP4⁺MAGE-A2-; FIG. 12B) or SK-MEL-37 (DP4⁺MAGE-A2+; FIG. 12C) and SK-MEL-28 (DP4- MAGE-A2⁺; FIG. 12D) and Me275 (DP4- MAGE-A2⁺; FIG. 12E) transduced with DP4. *, P<0.05 by Student's t-test. Bars and error bars represent the mean±SD of results in triplicate experiments. At least 2 independent experiments were performed.

FIGS. 13A-13Q are histograms comparing expression levels of wild-type HLADP*04:01 and derivatives thereof in K562 cells stained with the anti-HLA class II mAb clone 9-49. Open histograms represent the isotype control staining.

FIGS. 14A-14F provide data illustrating the enhanced CD4 binding ability of modified DQ molecules. FIG. 14A is a table comparing the amino acid sequences of DPB1*04:01, DQB1*05:01, and DQB1*05:01^{L114W/V143M+4reps}, with mutated amino acids underlined. FIGS. 14B and 14C are graphical representations of data of class II-deficient K562 cells stably expressing wild-type DQ5 (DQA1*01:01/DQB1*05:01), DQ5^L114W/V143M, DQ5^{L114W/V143M+4reps}, wild-type DP4, or DP4^L112W/V141Mstained with sCD4, as shown in FIG. 14A. FIG. 14D shows the CD4 binding ability of a series of K562 derivatives individually expressing DQ5^{L114W/V143M+4reps}mutants with a single amino acid reversal at one of the four positions, similarly stained with sCD4. FIG. 14E is a table listing the amino acid sequences of DPB1*04:01, DQB1*02:01, DQB1*04:02 and DQB1*06:01 with replaced amino acids underlined. Note that unlike DQB1*05:01, DQB1*02:01, DQB1*04:02 and DQB1*06:01 encode Val at position 116, similar to DPB1*04:01, which codes for Val at position 114. FIG. 14F provides graphical representations of data showing that the L114W/V143M+3reps replacements in the R chains enhanced the binding of DQ2, DQ4, and DQ6 to CD4. At least 2 independent experiments were performed. *, P<0.05 by Student's t-test. Bars and error bars represent the mean±SD of results in triplicate experiments.

FIGS. 15A-15B are graphical representations illustrating that affinity-matured DQ dimers detected cognate TCRs expressed in human primary CD4⁺ T cells. DQ5 (DQA1*01:01-DQB1*05:01)-restricted DDX3Y-specific TCR (E6) (FIG. 15A) and DQ6 (DQA1*01:02-DQB1*06:02)-restricted influenza virus HA-specific TCR (DM2) (FIG. 15B) were reconstituted in human primary CD4⁺ T cells and stained by DQ5^{L114W/V143M+4reps}and DQ6^{L114W/V143M+3reps}dimers, respectively. At least 2 independent experiments were performed.

FIGS. 16A-16Q are graphical representations of histograms illustrating the comparable expression levels of HLA class II genes. HLA-DQ and their derivatives were reconstituted in K562 cells and stained with anti-HLA class II monoclonal antibodies. The surface expression of each DQ2, DQ5, and DQ6 allele was detected using the anti-HLA class II monoclonal antibody clone 9-49(I3) (DQ5 and DQ6) or the anti-class II monoclonal antibody clone T639 (DQ2 and DQ4). Open histograms represent the isotype control staining.

FIGS. 17A-17F provide data illustrating the enhanced CD4 binding ability of modified DR molecules. FIG. 17A is a table comparing the amino acid sequences of DPB1*04:01, DRB1*01:01, and DRB1*01:01^{L114W/V143M+6reps}, with mutated amino acids underlined. FIGS. 17B and 17C are graphical representations of data of class II-deficient K562 cells stably transduced with wild-type DR1 (DRA1*01:01/DRB1*01:01), DR1^L114W/V143M, DR1^{L114W/V143M+6reps}, wild-type DP4, or DP4^L112W/V141Mand stained with sCD4. FIGS. 17D-17E show the CD4 binding ability of a series of K562 derivatives individually expressing DR1^{L114W/V143M+6reps}mutants with a single amino acid reversal at one of the six positions (FIG. 17D), similarly stained with sCD4 and DRB1^{L114W/V143M+2reps}, which carries S118H and T157I along with L114W/V143M (FIG. 17E). FIG. 17F is a table listing the amino acid sequences of DPB1*04:01 and DRB1 alleles of DR3, DR4, DR7, DR10, DR11, and DR13 were compared along with those of DRB1^{L114W/V143M+6reps}and DRB1^{L114W/V143M+2reps}, with mutated amino acids underlined. FIGS. 17G-17L are graphical representations of data showing that the L114W/V143M+2reps mutations enhanced the binding of DR3, DR4, DR7, DR10, DR11, and DR13 to CD4 better than the L114W/V143M+6reps mutations. At least 2 independent experiments were performed. *, P<0.05 by Student's t-test. Bars and error bars represent the mean±SD of results in triplicate experiments. FIGS. 17M-17N are biolayer interferometry sensorgrams showing the interaction of biotinylated HLA-DR1 (ligand) with soluble CD4 (analyte) over a range of concentrations. Binding experiments for wild-type DR1 (FIG. 17M) and DR1^{L114W/V143M+2reps}(FIG. 17N) were performed in parallel, and binding was not detected for wild-type DR1 (FIG. 17M). FIG. 17O is a graph showing the affinity between DR1^{L114W/V143M+2reps}and CD4 as quantified by steady-state analysis. All data are representative of two independent experiments.

FIGS. 18A-18D are graphical representations illustrating that affinity-matured DR dimers detected cognate TCRs are expressed in human primary CD4⁺ T cells. DR1-restricted TCRs (HA1.7 and SB95) (FIG. 18A), DR7-restricted TCR (SD334) (FIG. 18B) and DR11-restricted TCR (F24) (FIG. 18C) were reconstituted in primary human T cells and stained by respective DR^{L114W/V143M+2reps}dimers. DR11-restricted F24-transduced CD4⁺ T cells were stained with the DR11^{L114W/V143M+2reps}dimer and an anti-Vβ 22 mAb (FIG. 18D). Note that F24 expresses Vβ22. At least 2 independent experiments were performed.

FIGS. 19A-19D are drawings of model structures of HLA-DR1^{L114W/V143M+2reps}and the human CD4 complex. FIG. 19A provides an overview ribbon model of the ternary complex model structure of DRA1*01:01, DRB1*01:01, and CD4, as indicated. FIGS. 19B-19D provide close-up views of four mutated residues: L114W and V143M (FIG. 19B), S118H (FIG. 19C) and T157I (FIG. 19D) in wild-type DR1 (left) and mutated DR1^{L114W/V143M+2reps}(right), as illustrated using ball-and-stick representation.

FIGS. 20A-20II are graphical representations of histograms illustrating the comparable expression levels of HLA class II genes. HLA-DR and their derivatives were reconstituted in K562 cells and stained with anti-HLA class II monoclonal antibodies. The surface expression of all DR alleles was detected using the anti-HLA class II monoclonal antibody clone 9-49(I3). Open histograms represent the isotype control staining.

FIGS. 21A-21D are graphical representations of data showing comparison of DP4^L112W/V141Mdimers and dextramers for the staining of endogenous TRPC1_578-597-specific CD4⁺ T cells. Endogenous (non-transduced) TRPC1_578-597-specific CD4⁺ T cells were expanded from a melanoma patient by stimulation with peptide-pulsed and irradiated DP4⁺ artificial APCs and stained with DP4^L112W/V141MTRPC1_578-597dimers (FIG. 21B) or a TRPC1_578-597dextramer (FIG. 21D). The corresponding CLIP multimers were used as controls (FIGS. 21A and 21C).

FIGS. 22A-22F are graphical representations of data showing comparison of DP4^L112W/V141Mdimers and conventional DP4 tetramers and dextramers for the staining of endogenous NY-ESO-1_157-170-specific T cells. CD4⁺ T cells were purified from DP4⁺ healthy donor No. 4 and stimulated once with NY-ESO-1_157-170-pulsed and irradiated DP4⁺ artificial APCs. Expanded CD4⁺ T cells were individually stained as indicated by three different DP4 multimers (DP4^L112W/V141Mdimers (FIG. 22B), DP4 tetramers (FIG. 22D), or DP4 dextramers (FIG. 22F)).

FIGS. 23A-23Y are graphical representations of data showing pathogen-specific CD4⁺ T cells subjected to ex vivo staining with DP4^L112W/V141Mdimers. Memory CD4⁺ T cells were purified from five DP4⁺ donors and subjected to ex vivo staining with the DP4^L112W/V141Mdimers for the following pathogen-associated peptides without in vitro stimulation: TT_948-968(FIGS. 23F-23J), HSV-2-UL21_283-302(FIGS. 23K-230), Flu-HA527-546 (FIGS. 23P-23T), and RSV-GP_162-175(FIGS. 23U-23Y). The CLIP peptide was used as a negative control (FIGS. 23A-23E).

FIGS. 24A-24W are graphical representations of data showing endogenous RSV-GP_162-175-specific CD4⁺ T cell clones successfully established from DP4^L112W/V141Mdimer⁺ cells. Memory CD4⁺ T cells were purified from DP4⁺ Donor No. 06 and subjected to ex vivo staining with DP4^L112W/V141MRSV-GP_162-175dimers without in vitro stimulation. Dimer⁺CD4⁺ T cells were then cloned by limiting dilution. FIGS. 24A-24V are graphical representations of representative dimer staining data of 10 dimer-positive and 1 dimer-negative single-cell clones. Seventy-seven out of 84 clones (91.7%) were successfully stained with DP4^L112W/V141MRSV-GP_162-175dimers. FIG. 24W is a bar graph showing antigen-specific IL-2 production in RSV-GP_162-175dimer⁺single-cell clones.

FIGS. 25A-25S are graphical representations of data showing endogenous DP4 TT_948-968-specific CD4⁺ T cell clones successfully established from DP4^L112W/V141Mdimer⁺ cells. Memory CD4⁺T cells were purified from DP4⁺Donor No. 04 and subjected to ex vivo staining with DP4^L112W/V141MTT_948-968dimers without in vitro stimulation. Dimer⁺ CD4⁺ T cells were then cloned by limiting dilution. FIGS. 25A-25R are graphical representations of representative dimer staining data of 8 dimer-positive and 1 dimer-negative single-cell clones. Twenty-six out of 29 clones (89.7%) were successfully stained with DP4^L112W/V141MTT_948-968dimers. FIG. 25S is a bar graph showing antigen-specific IL-2 production in TT_948-968dimer⁺single-cell clones.

FIGS. 26A-26NN are graphical representations of DP4 multimer staining of RSV-GP (FIGS. 26A-26P) and TT (FIGS. 26O-26NN) dimer⁺single-cell clones. RSV-GP dimer⁺single-cell clones (c6, c12, c26, and c39) were stained with either DP4^L112W/V141MRSV-GP_162-175dimers (FIGS. 26B, 26D, 26F, and 26H) or wild-type DP4 dextramers (FIGS. 26J, 26L, 26N, and 26P). TT dimer⁺single-cell clones (c2, c4, c6, and c9) were individually stained with three different DP4 TT_948-968multimers (DP4^L112W/V141Mdimers (FIGS. 26R, 26T, 26V, and 26X), wild-type DP4 tetramers (FIGS. 26Z, 26BB, 26DD, and 26FF), and wild-type DP4 dextramers (FIGS. 26HH, 26JJ, 26LL, and 26NN).

FIGS. 27A-27L are graphical representations showing that DQ5^{L114W/V143M+4reps}dimers robustly stained E6-transduced CD4⁺ T cells. E6 was reconstituted in CD4⁺ T cells, which were then stained with wild-type DQ5 (FIGS. 27D and 27J), DQ5^L114W/V143M(FIGS. 27E and 27K), and DQ5^{L114W/V143M+4reps}(FIGS. 27F and 27L) CLIP control dimers (FIGS. 4D-4F) and dimers specific to DDX3Y_171-190(FIGS. 27J-27L). Control cells not transduced with a TCR are shown in FIGS. 27A-27C and 27G-27I.

FIGS. 28A-28H are graphical representations showing cloning of DQ5-restricted TCR using affinity matured dimer. Primary CD4⁺ T cells were purified from a DQ5.1⁺ melanoma patient and stimulated with irradiated GPC3_138-157-pulsed aAPCs expressing DQ5.1. Two weeks later, stimulated CD4⁺ T cells were stained with cognate GPC3_138-157-DQ5^{L114W/V143M+4reps}dimers (FIGS. 28A-28B). The GPC3 specific TCR was reconstituted in TCR-defective Jurkat 76/CD4 cells, and stained by the respective DQ5^{L114W/V143M+4reps}dimers (FIG. 28C (E6/Control); FIG. 28D (E6/GPC3_138-157); FIG. 28E (DQ5-06-GPC3_138-157/Control); and FIG. 28F (DQ5-06-GPC3_138-157/GPC3_138-157)). Jurkat 76/CD4 cells expressing the GPC3 specific TCR were stimulated by DQ5-K562 cells pulsed with the respective peptides in IL-2 ELISPOT assays (FIG. 28G).

FIGS. 29A-29L are graphical representations showing influenza virus hemagglutinin-specific peripheral CD4⁺ T cells subjected to ex vivo staining with DR1^{L114W/V143M+2reps}dimers. Memory CD4⁺ T cells were purified from two DR1⁺ donors (No. 07 (FIGS. 29A-29F) and No. 08 (FIGS. 29G-29L)) and stained with DR1^{L114W/V143M+2reps}dimers specific to Flu-HA_5-24(FIGS. 29B and 29H), Flu-HA_117-136(FIGS. 29C and 29I), Flu-HA_232-251(FIGS. 29D and 29J), Flu-HA_268-287(FIGS. 29E and 29K), and Flu-HA_306-318(FIGS. 29F and 29L) influenza virus hemagglutinin (Flu-HA) peptides without in vitro stimulation. The CLIP peptide was used as a negative control (FIGS. 29A and 29G).

FIGS. 30A-30X are graphical representations showing DR1^{L114W/V143M+6reps}and DR1^{L114W/V143M+2reps}dimers robustly stained HA1.7-transduced CD4⁺ T cells. HA1.7 was reconstituted in primary CD4⁺ T cells, which were then stained with wild-type DR1 (FIGS. 30I and 30M), DR1^L114W/V143M(FIGS. 30J and 30N), DR1^{L114W/V143M+6reps}(FIGS. 30K and 30O), and DR1^{L114W/V143M+2reps}(FIGS. 30L and 30P) dimers without a transduced TCR (FIGS. 30I-30L) and transduced with an HA1.7 TCR (FIGS. 30M-30P), with CLIP dimers used as negative controls (FIGS. 30A-30H). In addition, HA1.7 was reconstituted in primary CD4⁺ T cells, which were then stained with DR1^{L114W/V143M+2reps}dimer (FIGS. 30O-30T) or a wild-type DR1 dextramer (FIGS. 30U-30X) specific to Flu-HA_306-318.

FIGS. 31A-31P are graphical representations showing data for cloning of DR1-restricted TCRs using affinity matured dimer. Primary CD4⁺ T cells were purified from two DR1⁺ melanoma patients and stimulated with irradiated HSD17B12_225-244-pulsed (FIG. 31B) and LY6K_99-118-pulsed (FIG. 31D) aAPCs expressing DR1. Two weeks later, stimulated CD4⁺ T cells were stained with cognate DR1^{L114W/V143M+2reps}dimers (FIGS. 31A-31D). The DR1-restricted TCRs were reconstituted in primary CD4⁺ T cells, and stained by the respective dimer (FIGS. 31E-31M). Primary CD4⁺ T cells expressing the DR1-restricted DR1-07-HSD17B12_225-244(FIG. 31N) and DR1-08-LY6K_99-118(FIG. 31O) TCRs were stimulated by DR1-K562 cells pulsed with HSD17B12_225-244(FIG. 31N) and LY6K_99-118(FIG. 31O) peptides, respectively, in IL-2 ELISPOT assays.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure is directed to methods of identifying MHC class II-specific TCRs comprising contacting a T cell with a complex comprising an MHC class II molecule and a peptide, wherein the MHC class II molecule has a higher affinity for CD4 than a naturally occurring MHC class II molecule has for CD4. In some aspects, the MHC class II molecule comprises an alpha chain and a beta chain, wherein the beta chain of the MHC class II molecule comprises an amino acid sequence having one or more mutations relative to a wild-type beta chain of a MHC class II molecule.

L. Terms

In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower).

It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided.

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

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

“Administering” refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some aspects, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

The term “T cell receptor” (TCR), as used herein, refers to a heteromeric cell-surface receptor capable of specifically interacting with a target antigen. As used herein, “TCR” includes but is not limited to naturally occurring and non-naturally occurring TCRs; full-length TCRs and antigen binding portions thereof, chimeric TCRs; TCR fusion constructs; and synthetic TCRs. In human, TCRs are expressed on the surface of T cells, and they are responsible for T cell recognition and targeting of antigen presenting cells. Antigen presenting cells (APCs) display fragments of foreign proteins (antigens) complexed with the major histocompatibility complex (MHC; also referred to herein as complexed with an HLA molecule, e.g., an HLA class II molecule). A TCR recognizes and binds to the peptide:HLA complex and recruits CD8 (for MHC Class I molecules) or CD4 (for MHC class II molecules), activating the TCR. The activated TCR initiates downstream signaling and an immune response, including the destruction of the EPC.

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

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

Where not expressly stated, and unless the context indicates otherwise, the term “TCR” also includes an antigen-binding fragment or an antigen-binding portion of any TCR disclosed herein, and includes a monovalent and a divalent fragment or portion, and a single chain TCR. The term “TCR” is not limited to naturally occurring TCRs bound to the surface of a T cell. As used herein, the term “TCR” further refers to a TCR described herein that is expressed on the surface of a cell other than a T cell (e.g., a cell that naturally expresses or that is modified to express CD4, as described herein), or a TCR described herein that is free from a cell membrane (e.g., an isolated TCR or a soluble TCR).

An “antigen binding molecule,” “portion of a TCR,” or “TCR fragment” refers to any portion of an TCR less than the whole. An antigen binding molecule can include the antigenic CDRs.

An “antigen” refers to any molecule, e.g., a peptide, that provokes an immune response or is capable of being bound by a TCR. An “epitope,” as used herein, refers to a portion of a polypeptide that provokes an immune response or is capable of being bound by a TCR. The immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. A person of skill in the art would readily understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. An antigen and/or an epitope can be endogenously expressed, i.e. expressed by genomic DNA, or can be recombinantly expressed. An antigen and/or an epitope can be specific to a certain tissue, such as a diseased cell, e.g., a cancer cell, or it can be broadly expressed. In addition, fragments of larger molecules can act as antigens. In one aspect, antigens are tumor antigens. An epitope can be present in a longer polypeptide (e.g., in a protein), or an epitope can be present as a fragment of a longer polypeptide. In some aspects, an epitope is complexed with a major histocompatibility complex (MHC; also referred to herein as complexed with an HLA molecule, e.g., an HLA class 1 molecule).

The term “autologous” refers to any material derived from the same individual to which it is later to be re-introduced. For example, an autologous T cell therapy comprises administering to a subject a T cell that was isolated from the same subject. The term “allogeneic” refers to any material derived from one individual which is then introduced to another individual of the same species. For example, an allogeneic T cell transplantation comprises administering to a subject a T cell that was obtained from a donor other than the subject.

“CCND1,” “G1/S-specific cyclin-D1,” “B-cell lymphoma 1 protein,” “BCL-1,” or “PRAD1,” as used herein, refers to a human regulatory component of the cyclin D1-CDK4 (DC) complex that phosphorylates and inhibits members of the retinoblastoma (RB) protein family including RB1 and regulates the cell-cycle during G1/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complex and the subsequent transcription of E2F target genes which are responsible for the progression through the G1 phase. CCND1 is also involved in hypophosphorylation of RB1 in early G1 phase. Cyclin D-CDK4 complexes are major integrators of various mitogenenic and antimitogenic signals. CCND1 is also a substrate for SMAD3, phosphorylating SMAD3 in a cell-cycle-dependent manner and repressing its transcriptional activity. CCND1 is also a component of the ternary complex, cyclin D1/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex, and CCND1 exhibits transcriptional corepressor activity with INSM1 on the NEUROD1 and INS promoters in a cell cycle-independent manner. Mutations, amplification, and overexpression of CCND1, which alter cell cycle progression, are observed frequently in a variety of tumors and may contribute to tumorigenesis.

As used herein, CCND1 refers to not only the full-length canonical sequence, but also variants and fragments thereof. The amino acid sequence of CCND1 (SEQ ID NO: 27) is provided in Table 1A (UniProtKB-P24385).

TABLE 1A

CCND1 Amino Acid Sequence

CCND1 Amino Acid Sequence

MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCVQK

EVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQ

LLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELLLVNKLKW

NLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDVKFISNPP

SMVAAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQ

IEALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI(SEQ

ID NO: 27)

“MUC5AC” or “mucin 5AC,” as used herein, refers to a human gel-forming glycoprotein of gastric and respiratory tract epithelia that protects the mucosa from infection and chemical damage by binding to inhaled microorganisms and particles that are subsequently removed by the mucocilary system.

As used herein, MUC5AC refers to not only the full-length canonical sequence, but also variants and fragments thereof. The amino acid sequence of MUC5AC (SEQ TD NO: 28) is provided in Table 1B (UniProtKB-P98088).

TABLE 1B

MUC5AC Amino Acid Sequence

MUC5AC Amino Acid Sequence

MSVGRRKLALLWALALALACTRHTGHAQDGSSESSYKHHPALSPIARGPSGVPLRGATVFPSLRTIPVVRASNPAHN

GRVCSTWGSFHYKTFDGDVFRFPGLCNYVFSEHCGAAYEDFNIQLRRSQESAAPTLSRVLMKVDGVVIQLTKGSVLV

NGHPVLLPFSQSGVLIQQSSSYTKVEARLGLVLMWNHDDSLLLELDTKYANKTCGLCGDFKGMPVVSELLSHNTKLT

PMEFGNLQKMDDPTDQCQDPVPEPPRNCSTGFGICEELLHGQLFSGCVALVDVGSYLEACRQDLCFCEDTDLLSCVC

HTLAEYSRQCTHAGGLPQDWRGPDFCPQKCPNNMQYHECRSPCADTCSNQEHSRACEDHCVAGCFCPEGTVLDDIGQ

TGCVPVSKCACVYNGAAYAPGATYSTDCTNCTCSGGRWSCQEVPCPGTCSVLGGAHFSTFDGKQYTVHGDCSYVLTK

PCDSSAFTVLAELRRCGLTDSETCLKSVTLSLDGAQTVVVIKASGEVFLNQIYTQLPISAANVTIFRPSTFFIIAQT

SLGLQLNLQLVPTMQLFMQLAPKLRGQTCGLCGNFNSIQADDFRTLSGVVEATAAAFFNTFKTQAACPNIRNSFEDP

CSLSVENEKYAQHWCSQLTDADGPFGRCHAAVKPGTYYSNCMFDTCNCERSEDCLCAALSSYVHACAAKGVQLGGWR

DGVCTKPMTTCPKSMTYHYHVSTCQPTCRSLSEGDITCSVGFIPVDGCICPKGTFLDDTGKCVQASNCPCYHRGSMI

PNGESVHDSGAICTCTHGKLSCIGGQAPAPVCAAPMVFFDCRNATPGDTGAGCQKSCHTLDMTCYSPQCVPGCVCPD

GLVADGEGGCITAEDCPCVHNEASYRAGQTIRVGCNTCTCDSRMWRCTDDPCLATCAVYGDGHYLTFDGQSYSFNGD

CEYTLVQNHCGGKDSTQDSFRVVTENVPCGTTGTTCSKAIKIFLGGFELKLSHGKVEVIGTDESQEVPYTIRQMGIY

LVVDTDIGLVLLWDKKTSIFINLSPEFKGRVCGLCGNFDDIAVNDFATRSRSVVGDVLEFGNSWKLSPSCPDALAPK

DPCTANPFRKSWAQKQCSILHGPTFAACHAHVEPARYYEACVNDACACDSGGDCECFCTAVAAYAQACHEVGLCVSW

RTPSICPLFCDYYNPEGQCEWHYQPCGVPCLRTCRNPRGDCLRDVRGLEGCYPKCPPEAPIFDEDKMQCVATCPTPP

LPPRCHVHGKSYRPGAVVPSDKNCQSCLCTERGVECTYKAEACVCTYNGQRFHPGDVIYHTTDGTGGCISARCGANG

TIERRVYPCSPTTPVPPTTFSFSTPPLVVSSTHTPSNGPSSAHTGPPSSAWPTTAGTSPRTRLPTASASLPPVCGEK

CLWSPWMDVSRPGRGTDSGDFDTLENLRAHGYRVCESPRSVECRAEDAPGVPLRALGQRVQCSPDVGLTCRNREQAS

GLCYNYQIRVQCCTPLPCSTSSSPAQTTPPTTSKTTETRASGSSAPSSTPGTVSLSTARTTPAPGTATSVKKTFSTP

SPPPVPATSTSSMSTTAPGTSVVSSKPTPTEPSTSSCLQELCTWTEWIDGSYPAPGINGGDFDTFQNLRDEGYTFCE

SPRSVQCRAESFPNTPLADLGQDVICSHTEGLICLNKNQLPPICYNYEIRIQCCETVNVCRDITRLPKTVATTRPTP

HPTGAQTQTTFTTHMPSASTEQPTATSRGGPTATSVTQGTHTTLVTRNCHPRCTWTKWFDVDFPSPGPHGGDKETYN

NIIRSGEKICRRPEEITRLQCPAKSHPEVSIEHLGQVVQCSREEGLVCRNQDQQGPFKMCLNYEVRVLCCETPRGCH

MTSTPGSTSSSPAQTTPSTTSKTTETQASGSSAPSSTPGTVSLSTARTTPAPGTATSVKKTFSTPSPPPVPATSTSS

MSTTAPGTSVVSSKPTPTEPSTSSCLQELCTWTEWIDGSYPAPGINGGDFDTFQNLRDEGYTFCESPPSVQCRAESF

PNTPLADLGQDVICSHTEGLICLNKNQLPPICYNYEIRIQCCETVNVCRDITRPPKTVATTRPTPHPTGAQTQTTFT

THMPSASTEQPTATSRGGPTATSVTQGTHTTPVTRNCHPRCTWTTWFDVDFPSPGPHGGDKETYNNIIRSGEKICRR

PEEITRLQCRAKSHPEVSIEHLGQVVQCSREEGLVCRNQDQQGPFKMCLNYEVRVLCCETPKGCPVTSTPVTAPSTP

SGRATSPTQSTSSWQKSRTTTLVTTSTTSTPQTSTTYAHTTSTTSAPTARTTSAPTTRTTSASPASTTSGPGNTPSP

VPTTSTISAPTTSITSAPTTSTTSAPTSSTTSGPGTTPSPVPTTSITSAPTTSTTSAPTTSTTSARTSSTTSATTTS

RISGPETTPSPVPTTSTTSATTTSTTSAPTTSTTSAPTSSTTSSPQTSTTSAPTTSTTSGPGTTPSPVPTTSTTSAP

TTRTTSAPKSSTTSAATTSTTSGPETTPRPVPTTSTTSSPTTSTTSAPTTSTTSASTTSTTSGAGTTPSPVPTTSTT

SAPTTSTTSAPISSTTSATTTSTTSGPGTTPSPVPTTSTTSAPTTSTTSGPGTTPSAVPTTSITSAPTTSTNSAPIS

STTSATTTSRTSGPETTPSPVPTASTTSASTTSTTSGPGTTPSPVPTTSTISVPTTSTTSASTTSTTSASTTSTTSG

PGTTPSPVPTTSTTSAPTTSTTSAPTTSTISAPTTSTTSATTTSTTSAPTPPRTSAPTTSTISASTTSTTSATTTST

TSATTTSTISAPTTSTTLSPTTSTTSTTITSTTSAPISSTTSTPQTSTTSAPTTSTTSGPGTTSSPVPTTSTTSAPT

TSTTSAPTTRTTSVPTS3TTSTATTSTTSGPGTTPSPVPTTSTTSAPTTRTTSAPTTSTTSAPTTSTTSAPTSSTTS

ATTTSTISVPTTSTTSVPGTTPSPVPTTSTISVPTTSTTSASTTSTTSGPGTTPSPVPTTSTTSAPTTSTTSAPTTS

TISAPTTSTPSAPTTSTTLAPTTSTTSAPTTSTTSTPTSSTTSSPQTSTTSASTTSITSGPGTTPSPVPTTSTTSAP

TTSTTSAATTSTISAPTTSTTSAPTTSTTSASTASKTSGLGTTPSPIPTTSTTSPPTTSTTSASTASKTSGPGTTPS

PVPTTSTIFAPRTSTTSASTTSTTPGPGTTPSPVPTTSTASVSKTSTSHVSISKTTHSQPVTRDCHLRCTWTKWFDI

DFPSPGPHGGDKETYNNIIRSGEKICRRPEEITRLQCRAESHPEVSIEHLGQVVQCSREEGLVCRNQDQQGPFKMCL

NYEVPVLCCETPKGCPVTSTPVTAPSTPSGRATSPTQSTSSWQKSRTTTLVTTSTTSTPQTSTTSAPTTSTTSAPTT

STTSAPTTSTTSTPQTSISSAPTSSTTSAPTSSTISARTTSIISAPTTSTTSSPTTSTTSATTTSTTSAPTSSTTST

PQTSKTSAATSSTTSGSGTTPSPVTTTSTASVSKTSTSHVSVSKTTHSQPVTRDCHPRCTWTKWFDVDFPSPGPHGG

DKETYNNIIRSGEKICRRPEEITRLQCRAKSHPEVSIEHLGQVVQCSREEGLVCRNQDQQGPFKMCLNYEVRVLCCE

TPKGCPVTSTSVTAPSTPSGRATSPTQSTSSWQKSRTTTLVTSSITSTTQTSTTSAPTTSTTPASIPSTTSAPTTST

TSAPTTSTTSAPTTSTTSTPQTTTSSAPTSSTTSAPTTSTISAPTTSTISAPTTSTTSAPTASTTSAPTSTSSAPTT

NTTSAPTTSTTSAPITSTISAPTTSTTSTPQTSTISSPTTSTTSTPQTSTTSSPTTSTTSAPTTSTTSAPTTSTTST

PQTSISSAPTSSTTSAPTASTISAPTTSTTSFHTTSTTSPPTSSTSSTPQTSKTSAATSSTTSGSGTTPSPVPTTST

ASVSKTSTSHVSVSKTTHSQPVTRDCHPRCTWTKWEDVDFPSPGPHGGDKETYNNIIRSGEKICRRPEEITRLQCRA

ESHPEVSIEHLGQVVQCSREEGLVCRNQDQQGPFKMCLNYEVRVLCCETPKGCPVTSTPVTAPSTPSGRATSPTQST

SSWQKSRTTTLVTTSTTSTPQTSTTSAPTTSTIPASTPSTTSAPTTSTTSAPTTSTTSAPTHRTTSGPTTSTTLAPT

TSTTSAPTTSTNSAPTTSTISASTTSTTSAPTTSTISSPTSSTTSTPQTSKTSAATSSTTSGSGTTPSPVPTTSTTS

ASTTSTTSAPTTSTTSGPGTTPSPVPSTSTTSAATTSTTSAPTTRTTSAPTSSMTSGPGTTPSPVPTTSTTSAPTTS

TTSGPGTTPSPVPTTSTTSAPITSTTSGPGSTPSPVPTTSTTSAPTTSTTSASTASTTSGPGTTPSPVPTTSTTSAP

TTRTTSASTASTTSGPGSTPSPVPTTSTTSAPTTRTTPASTASTTSGPGTTPSPVPTTSTTSASTTSTISLPTTSTT

SAPITSMTSGPGTTPSPVPTTSTTSAPTTSTTSASTASTTSGPGTTPSPVPTTSTTSAPTTSTTSASTASTTSGPGT

SLSPVPTTSTTSAPTTSTTSGPGTTPSPVPTTSTTSAPTTSTTSGPGTTPSPVPTTSTTPVSKTSTSHLSVSKTTHS

QPVTSDCHPLCAWTKWFDVDFPSPGPHGGDKETYNNIIRSGEKICRRPEEITRLQCRAESHPEVNIEHLGQVVQCSR

EEGLVCRNQDQQGPFKMCLNYEVRVLCCETPRGCPVTSVTPYGTSPTNALYPSLSTSMVSASVASTSVASSSVASSS

VAYSTQTCFCNVADRLYPAGSTIYRHRDLAGHCYYALCSQDCQVVRGVDSDCPSTTLPPAPATSPSISTSEPVTELG

CPNAVPPRKKGETWATPNCSEATCEGNNVISLRPRTCPRVEKPTCANGYPAVKVADQDGCCHHYQCQCVCSGWGDPH

YITFDGTYYTFLDNCTYVLVQQIVPVYGHFRVLVDNYFCGAEDGLSCPRSIILEYHQDRVVLTRKPVHGVMTNEIIF

NNKVVSPGFRKNGIVVSRIGVKMYATIPELGVQVMFSGLIFSVEVPFSKFANNTEGQCGTCTNDRKDECRTPRGTVV

ASCSEMSGLWNVSIPDCPACHRPHPTPTTVGPTTVGSTTVGPTTVGSTTVGPTTPPAPCLPSPICQLILSKVFEPCH

TVIPPLLEYEGCVFDRCHMTDLDVVCSSLELYAALCASHDICIDWRGRTGHMCPFTCPADKVIOPCGPSNPSYCYGN

DSASLGALPEAGPITEGCFCPEGMTLFSTSAQVCVPTGCPRCLGPHGEPVKVGHTVGMDCQECTCEAATWTLTCRPK

LCPLPPACPLPGFVPVPAPPQAGQCCPQYSCACNTSRCPAPVGCPEGARAIPTYQEGACCPVQNCSWTVCSINGTLY

QPGAVVSSSLCETCRCELPGGPPSDAFVVSCETQICNTHCPVGFEYQEQSGQCCGTCVQVACVTNTSKSPAHLFYPG

ETWSDAGNHCVTHQCEKHQDGLVVVTTKKACPPLSCSLDEARMSKDGCCRECPPPPPPYQNQSTCAVYHRSLIIQQQ

GCSSSEPVRLAYCRGNCGDSSSMYSLEGNTVEHRCQCCQELRTSLRNVTLHCTDGSSRAFSYTEVEECGCMGRRCPA

PGDTCHSEEAEPEPSQEAESGSWERGVPVSPMH (SEQ ID NO: 28)

“MAGE-A2,” “melanoma-associated antigen 2,” or “cancer/testis antigen 1.2,” as used herein, refers to a human protein primarily expressed by tumor cells. MAGE-A2 reduces p53/TP53 transactivation function through recruitment of HDAC3 to p53/TP53 transcription sites. MAGE-A2 represses p73/TP73 activity. In vitro, MAGE-A2 promotes cell viability in melanoma cell lines. MAGE-A2 is expressed in many tumors of several types, such as melanoma, head and neck squamous cell carcinoma, lung carcinoma, and breast carcinoma. However, in healthy tissue, MAGE-A2 is only expressed in the testes.

As used herein, MAGE-A2 refers to not only the full-length sequence, but also variants and fragments thereof. The amino acid sequence of MAGE-A2 (SEQ TD NO: 29) is provided in Table 1C (UniProtKB-P43356).

TABLE 1C

MAGE-A2 Amino Acid Sequence

MAGE-A2 Amino Acid Sequence

MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQQTASSSSTLVEVTL

GEVPAADSPSPPHSPQGASSFSTTINYTLWRQSDEGSSNQEEEGPRMFPD

LESEFQAAISRKMVELVHFLLLKYRAREPVTKAEMLESVLRNCQDFFPVI

FSKASEYLQLVFGIEVVEVVPISHLYILVTCLGLSYDGLLGDNQVMPKTG

LLIIVLAIIAIEGDCAPEEKIWEELSMLEVFEGREDSVFAHPRKLLMQDL

VQENYLEYRQVPGSDPACYEFLWGPRALIETSYVKVLHHTLKIGGEPHIS

YPPLHERALREGEE (SEQ ID NO: 29)

The term “HLA,” as used herein, refers to the human leukocyte antigen. HLA genes encode the major histocompatibility complex (MHC) proteins in humans. MHC proteins are expressed on the surface of cells, and are involved in activation of the immune response. HLA class II genes encode MHC class II proteins which are expressed on the surface of professional antigen presenting cells (APCs). Non-limiting examples of professional APCs include monocytes, macrophages, dendritic cells (DCs), and B lymphocytes. Some endothelial and epithelial cells can also express MHC class II molecules after inflammatory signals are activated. Humans lacking functional MHC class II molecules are extremely susceptible to an array of infectious diseases and typically die at a young age.

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

Both the MHC Class II alpha and beta chains are encoded by the HLA gene complex. The HLA complex is located within the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes of diverse function. The HLA gene complex is highly variant, with over 20,000 HLA alleles and related alleles, including over 250 MHC class II alpha chain alleles and 5,000 MHC class II beta chain alleles, known in the art, encoding thousands of MHC class II proteins (see, e.g., hla.alleles.org, last visited May 20, 2019, which is incorporated by reference herein in its entirety). For example one such HLA-DP allele, DP4 is the most frequently found allele in many ethnic groups.

Three loci in the HLA complex encode MHC Class II proteins: HLA-DP, HLA-DQ, and HLA-DR. HLA-DO and HLA-DM encode proteins that associate with the MHC class II molecule and support its configuration and function.

When the MHC class II molecule is complexed with an antigen peptide, the 10-30 amino acid long antigen peptide binds the peptide-binding groove and is presented extracellularly to CD4⁺cells. Both the alpha- and beta-chains fold into two separate domains; alpha-1 and alpha-2 for the alpha polypeptide, and beta-1 and beta-2 for the beta polypeptide. The open-ended peptide-binding groove which holds the presented antigen is found between the alpha-1 and beta-1 domains. Upon interaction with a CD4⁺T cell, the MHC class II complex interacts with a T cell receptor (TCR) expressed on the surface of the T cell. In addition, the beta chain of the MHC class II molecule weakly interacts (K_D>2 mM) with CD4 expressed on the surface of the T cell. The canonical CD4 amino acid sequence (UniProt-P01730) is provided in Table 2 (SEQ ID NO: 10).

TABLE 2

Human CD4 Amino Acid Sequence

MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQ

FHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLK

IEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSS

PSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIV

VLAFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKS

WITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLA

LEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAK

VSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALI

VLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHR

FQKTCSPI (SEQ ID NO: 10)

A “cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A “cancer” or “cancer tissue” can include a tumor. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system including lymphoma, leukemia, and other leukocyte malignancies. In some aspects, the methods of the present invention can be used to reduce the tumor size of a tumor derived from, for example, bone cancer, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or intraocular malignant melanoma, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B cell malignancies, and combinations of said cancers. The particular cancer can be responsive to chemo- or radiation therapy or the cancer can be refractory.

A refractory cancer refers to a cancer that is not amendable to surgical intervention, and the cancer is either initially unresponsive to chemo- or radiation therapy or the cancer becomes unresponsive over time.

An “anti-tumor effect” as used herein, refers to a biological effect that can present as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or amelioration of various physiological symptoms associated with the tumor. An anti-tumor effect can also refer to the prevention of the occurrence of a tumor, e.g., a vaccine.

The term “progression-free survival,” which can be abbreviated as PFS, as used herein refers to the time from the treatment date to the date of disease progression per the revised IWG Response Criteria for Malignant Lymphoma or death from any cause.

“Disease progression” or “progressive disease,” which can be abbreviated as PD, as used herein, refers to a worsening of one or more symptom associated with a particular disease. For example, disease progression for a subject afflicted with a cancer can include an increase in the number or size of one or more malignant lesions, tumor metastasis, and death.

The “duration of response,” which can be abbreviated as DOR, as used herein refers to the period of time between a subject's first objective response to the date of confirmed disease progression, per the revised IWG Response Criteria for Malignant Lymphoma, or death.

The term “overall survival,” which can be abbreviated as OS, is defined as the time from the date of treatment to the date of death.

A “cytokine,” as used herein, refers to a non-antibody protein that is released by one cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. A cytokine can be endogenously expressed by a cell or administered to a subject. Cytokines may be released by immune cells, including macrophages, B cells, T cells, and mast cells to propagate an immune response. Cytokines can induce various responses in the recipient cell. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, and pro-inflammatory cytokines can promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase-proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

“Chemokines” are a type of cytokine that mediates cell chemotaxis, or directional movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), gamma-induced protein 10 (IP-10), and thymus and activation regulated chemokine (TARC or CCL17).

Other examples of analytes and cytokines of the present invention include, but are not limited to chemokine (C-C motif) ligand (CCL) 1, CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocyte chemoattractant protein 2 (MCP-2 or CCL8), CCL13, IL-1, IL-3, IL-9, IL-11, IL-12, IL-14, IL-17, IL-20, IL-21, granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), oncostatin M (OSM), CD154, lymphotoxin (LT) beta, 4-1BB ligand (4-1BBL), a proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-related ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF- and ApoL-related leukocyte-expressed ligand 1 (TALL-1), or TNF-related apoptosis-inducing ligand (TRAIL).

A “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

The term “infection,” as used herein refers to any type of invasion of one or more tissue of the body by a foreign agent. The term “infection” includes without limitation infection by a virus (including viroids and prions), a bacterium, a fungus, a parasite, and any combination thereof.

The term “lymphocyte” as used herein includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell toxic) lymphocyte that represent a major component of the inherent immune system. NK cells reject tumors and cells infected by viruses. It works through the process of apoptosis or programmed cell death. They were termed “natural killers” because they do not require activation in order to kill cells. T-cells play a major role in cell-mediated-immunity (no antibody involvement). T-cell receptors (TCR) differentiate T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the T cell's maturation. There are six types of T-cells, namely: Helper T-cells (e.g., CD4⁺cells), Cytotoxic T-cells (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8⁺T-cells or killer T cell), Memory T-cells ((i) stem memory T_SCMcells, like naive cells, are CD45RO−, CCR7⁺, CD45RA+, CD62L+(L-selectin), CD27⁺, CD28⁺ and IL-7Rα+, but they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells); (ii) central memory T_CMcells express L-selectin and the CCR7, they secrete IL-2, but not IFNγ or IL-4, and (iii) effector memory T_EMcells, however, do not express L-selectin or CCR7 but produce effector cytokines like IFNγ and IL-4), Regulatory T-cells (Tregs, suppressor T cells, or CD4⁺CD25⁺regulatory T cells), Natural Killer T-cells (NKT) and Gamma Delta T-cells. B-cells, on the other hand, play a principal role in humoral immunity (with antibody involvement). A B cell makes antibodies and antigens and performs the role of antigen-presenting cells (APCs) and turns into memory B-cells after activation by antigen interaction. In mammals, immature B-cells are formed in the bone marrow, where its name is derived from.

The terms “modified” and “mutated,” when used herein to refer to a nucleotide or amino acid sequence, refers to a change in the sequence relative to a wild-type sequence or a specified reference sequence. The terms “modified” and “mutated” do not require a step in a process for making the modified or mutated sequence (e.g., the modified beta chain sequence), unless otherwise specified. Rather, these terms indicate that there is a variation in the modified or mutated sequence relative to a reference sequence, e.g., a wild-type sequence. For example, a DP beta chain comprising a substitution mutation at a position corresponding to amino acid residue 112 of SEQ ID NO: 1 does not require that a wild-type DP beta chain has been physically altered to arrive at the recited DP beta chain; but rather that, when properly aligned, the recited DP beta chain comprises an amino acid residue at the recited position (residue 112) that is different from the amino acid residue at the corresponding position in a wild-type or reference DP beta chain.

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

As used herein “at a position corresponding to” is used as a means to identify a particular amino acid residue, e.g., a specific amino acid position, in a polynucleotide or a particular nucleic acid, e.g., a specific nucleic acid position, in a polypeptide. The position can be determined by properly aligning the sequence in question with the referenced sequence. A person of skill in the art would readily understand how to align to sequences to determine the relative position. For example, various alignment tools are available online, including, without limitation, “Clustal Omega Multiple Sequence Alignment,” available at www.ebi.ac.uk (last visited May 25, 2019).

The term “genetically engineered” or “engineered” refers to a method of modifying the genome of a cell, including, but not limited to, deleting a coding or non-coding region or a portion thereof or inserting a coding region or a portion thereof. In some aspects, the cell that is modified is a lymphocyte, e.g., a T cell or a modified cell that expresses CD4, which can either be obtained from a patient or a donor. The cell can be modified to express an exogenous construct, such as, e.g., a T cell receptor (TCR) disclosed herein, which is incorporated into the cell's genome. In some aspects, the cell is modified to express CD4.

An “immune response” refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

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

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

A “patient” as used herein includes any human who is afflicted with a cancer (e.g., a lymphoma or a leukemia). The terms “subject” and “patient” are used interchangeably herein.

The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

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

The terms “conditioning” and “pre-conditioning” are used interchangeably herein and indicate preparing a patient in need of a T cell therapy for a suitable condition. Conditioning as used herein includes, but is not limited to, reducing the number of endogenous lymphocytes, removing a cytokine sink, increasing a serum level of one or more homeostatic cytokines or pro-inflammatory factors, enhancing an effector function of T cells administered after the conditioning, enhancing antigen presenting cell activation and/or availability, or any combination thereof prior to a T cell therapy. In one aspect, “conditioning” comprises increasing a serum level of one or more cytokines, e.g., interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10), interleukin 5 (IL-5), gamma-induced protein 10 (IP-10), interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), placental growth factor (PLGF), C-reactive protein (CRP), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), or any combination thereof. In another aspect, “conditioning” comprises increasing a serum level of IL-7, IL-15, IP-10, MCP-1, PLGF, CRP, or any combination thereof.

“Treatment” or “treating” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease. In one aspect, “treatment” or “treating” includes a partial remission. In another aspect, “treatment” or “treating” includes a complete remission.

The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles “a” or “an” should be understood to refer to “one or more” of any recited or enumerated component.

The terms “about” or “comprising essentially of” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “comprising essentially of” can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” or “comprising essentially of” can mean a range of up to 10% (i.e., ±10%). For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (for 10%). Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of “about” or “comprising essentially of” should be assumed to be within an acceptable error range for that particular value or composition.

As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

Various aspects of the invention are described in further detail in the following subsections.

II. Methods of the Disclosure

The present disclosure is directed to methods of identifying MHC class II-specific TCRs comprising contacting a T cell with a complex comprising (i) an HLA class II molecule with enhanced CD4 binding and (ii) a peptide, e.g., an epitope. In certain aspects, the T cell expresses CD4. In certain aspects, the T cell expresses one or more TCRs. In some aspects, the MHC class II-specific TCR specifically binds the complex comprising the MHC class II molecule and the peptide.

In some aspects, the MHC class II molecule comprises an alpha chain and a beta chain, wherein the alpha chain, the beta chain, or both the alpha chain and the beta chain comprises an amino acid sequence having one or more mutations relative to a wild-type alpha chain and/or beta chain of a MHC class II molecule. In some aspects, the alpha chain comprises an amino acid sequence having one or more mutations relative to a wild-type alpha chain of a MHC class II molecule. In some aspects, the beta chain comprises an amino acid sequence having one or more mutations relative to a wild-type beta chain of a MHC class II molecule. In some aspects, the alpha chain comprises an amino acid sequence having one or more mutations relative to a wild-type alpha chain of a MHC class II molecule, and the beta chain comprises an amino acid sequence having one or more mutations relative to a wild-type beta chain of a MHC class II molecule.

In some aspects, the one or more mutations comprises a substitution mutation. In some aspects, the one or more mutations comprises a deletion mutation. In some aspects, the one or more mutations comprises an insertion mutation. In some aspects, the one or more mutations comprises a substitution of a single amino acid with one or more heterologous amino acids. In some aspects, the one or more mutations comprises the substitution of a single amino acid with a different amino acid. In some aspects, the one or more mutations comprises the substation of a single amino acid with 2 different amino acids, 3 different amino acids, 4 different amino acids, 5 different amino acids, or more than 5 different amino acids.

In some aspects, the MHC class II molecule is a dimer. In some aspects, the MHC class II molecule is a trimer. In some aspects, the MHC class II molecule is a tetramer.

Certain aspects of the present disclosure are directed to methods of enriching a target population of T cells obtained from a human subject. In some aspects, the method comprises contacting the T cells with an HLA class II molecule disclosed herein. In some aspects, the method comprises contacting the T cells with a cell, e.g., an APC, disclosed herein. In some aspects, following the contacting, the enriched population of T cells comprises a higher number of T cells capable of binding the HLA class II molecule relative to the number of T cells capable of binding the HLA class II molecule prior to the contacting.

Some aspects of the present disclosure are directed to a method of selecting a T cell capable of targeting a diseased cell, e.g., a tumor cell. In some aspects, the method comprises contacting a population of isolated T cells in vitro with a complex comprising an MHC class II molecule disclosed herein and a fragment of a polypeptide, e.g. an antigen expressed by a diseased cell, e.g., a tumor-expressed polypeptide, e.g., an epitope.

In some aspects, the T cells used in the methods disclosed herein are obtained from a human subject. The T cells obtained from the human subject can be any T cells disclosed herein. In some aspects, the T cells obtained from the human subject are tumor infiltrating lymphocytes (TIL).

In some aspects, the method further comprises selecting the T cell that is bound by the MHC class II molecule. In some aspects, the method further comprises administering to the human subject the enriched T cells. In some aspects, the subject is preconditioned prior to receiving the T cells, as described herein.

In some aspects, the method further comprises isolating the TCR that is bound to the MHC class II molecule. In some aspects, the method further comprises sequencing the TCR. In some aspects, the method further comprises cloning the TCR. In some aspects, the method further comprises recombinantly expressing the TCR, or a modified variant thereof, in a host cell. In some aspects, the host cell is an immune cell, e.g., a T cell. In some aspects, the method further comprises administering the host cell to a subject. In some aspects, the subject has a cancer, and the host cell comprising the TCR treats the cancer in the subject.

II.A. MHC Class II Molecules

The human leukocyte antigen (HLA) system (the major histocompatibility complex [MHC] in humans) is an important part of the immune system and is controlled by genes located on chromosome 6. It encodes cell surface molecules specialized to present antigenic peptides to the TCR on T cells. (See also Overview of the Immune System.) MHC molecules that present antigen (Ag) are divided into 2 main classes: Class I MHC molecules and Class II MHC molecules.

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

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

II.A.1. HLA-DP Molecules

Many HLA-DP alleles are known in the art, and any of the known alleles can be used in the methods of present disclosure. Examples of HLA-DP alpha chain and beta chain alleles are shown in Table 3. An updated list of HLA alleles is available at hla.alleles.org/(last visited on Feb. 27, 2019).

TABLE 3

DP Beta chain and alpha chain amino acid and nucleotide sequences.

Beta Chain

DPB1*04: 01 Extracellular Domain (SEQ ID NO: 1)

RATPENYLFQGRQECYAFNGTQRFLERYIYNREEFARFDSDVGEFRAVTELGRPAAEYWNSQKDILEEKRAVPD

RMCRHNYELGGPMTLQRRVQPRVNVSPSKKGPLQHHNLLVCHVTDFYPGSIQVRWELNGQEETAGVVSTNLIRN

GDWTFQILVMLEMTPQQGDVYTCQVEHTSLDSPVTVEWKAQSDSARSK

DPB1*04: 01 Extracellular Domain (SEQ ID NO: 2)

AGGGCCACTCCAGAGAATTACCTTTTCCAGGGACGGCAGGAATGCTACGCGTTTAATGGGACACAGCGCTTCCT

GGAGAGATACATCTACAACCGGGAGGAGTTCGCGCGCTTCGACAGCGACGTGGGGGAGTTCCGGGCGGTGACGG

AGCTGGGGCGGCCTGCTGCGGAGTACTGGAACAGCCAGAAGGACATCCTGGAGGAGAAGCGGGCAGTGCCGGAC

AGGATGTGCAGACACAACTACGAGCTGGGCGGGCCCATGACCCTGCAGCGCCGAGTCCAGCCTAGGGTGAATGT

TTCCCCCTCCAAGAAGGGGCCCTTGCAGCACCACAACCTGCTTGTCTGCCACGTGACGGATTTCTACCCAGGCA

GCATTCAAGTCCGATGGTTCCTGAATGGACAGGAGGAAACAGCTGGGGTCGTGTCCACCAACCTGATCCGTAAT

GGAGACTGGACCTTCCAGATCCTGGTGATGCTGGAAATGACCCCCCAGCAGGGAGATGTCTACACCTGCCAAGT

GGAGCACACCAGCCTGGATAGTCCTGTCACCGTGGAGTGGAAGGCACAGTCTGATTCTGCCCGGAGTAAG

DPB1*04: 01 L112W/V141M Extracellular Domain (SEQ ID NO: 3)

RATPENYLFQGRQECYAFNGTQRFLERYIYNREEFARFDSDVGEFRAVTELGRPAAEYWNSQKDILEEKRAVPD

RMCRHNYELGGPMTLQRRVQPRVNVSPSKKGPLQHHNWLVCHVTDFYPGSIQVRWFLNGQEETAGVMSTNLIRN

GDWTFQILVMLEMTPQQGDVYTCQVEHTSLDSPVTVEWKAQSDSARSK

Signal Peptide; DPB1*04: 01 L112W/V141M Extracellular Domain; Gly/Ser

Linker; Zip Sequences and His tag sequences) (SEQ ID NO: 4)

MMRPIVLVLLFATSALA
RATPENYLFQGRQECYAFNGTQRFLERYIYNREEFARFDSDVGEFRAVTELGRPAAE

YWNSQKDILEEKRAVPDRMCRHNYELGGPMTLQRRVQPRVNVSPSKKGPLQHHNWLVCHVTDFYPGSIQVRWFL

NGQEETAGVMSTNLIRNGDWTFQILVMLEMTPQQGDVYTCQVEHTSLDSPVTVEWKAQSDSARSK
GGGGSLEIE

AAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK

Full-length wild-type DPB1*04: 01 (SEQ ID NO: 5)

MMVLQVSAAPRTVALTALLMVLLTSVVQGRATPENYLFQGRQECYAFNGTQRFLERYIYNREEFARFDSDVGEF

RAVTELGRPAAEYWNSQKDILEEKRAVPDRMCRHNYELGGPMTLQRRVQPRVNVSPSKKGPLQHHNLLVCHVTD

FYPGSIQVRWFLNGQEETAGVVSTNLIRNGDWTFQILVMLEMTPQQGDVYTCQVEHTSLDSPVTVEWKAQSDSA

RSKTLTGAGGFVLGLIICGVGIFMHRRSKKVQRGSA

Signal Peptide; DPB1*04: 01 Extracellular Domain; and Gly/Ser Linker, Zip

Sequences, GS linker, and biotinylation sequences) (SEQ ID NO: 256)

MMRPIVLVLLFATSALA
RATPENYLFQGRQECYAFNGTQRFLERYIYNREEFARFDSDVGEFRAVTELGRPAAE

YWNSQKDILEEKRAVPDRMCRHNYELGGPMTLQRRVQPRVNVSPSEKGPLQHHNLLVCHVTDFYPGSIQVRWFL

NGQEETAGVVSTNLIRNGDWTFQILVMLEMTPQQGDVYTCQVEHTSLDSPVTVEWEAQSDSARSK
GGGGSLEIE

AAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGKGSGLNDIFEAQKIEWHE

Signal Peptide; DPB1*04: 01 Extracellular Domain; Gly/Ser Linker; Zip

Sequences; GSlinker and biotinylation sequences) (SEQ ID NO: 257)

ATGATGAGGCCCATCGTGCTGGTGCTGCTGTTCGCCACATCTGCCCTGGCC
AGAGCCACCCCCGAGAACTACCT

GTTTCAGGGCCGGCAGGAATGCTACGCCTTCAACGGCACCCAGCGGTTTCTGGAACGGTACATCTACAACCGGG

AAGAGTTCGCCAGATTCGACAGCGACGTGGGCGAGTTCAGAGCCGTGACAGAGCTGGGCAGACCTGCCGCCGAG

TACTGGAACAGCCAGAAGGACATCCTGGAAGAGAAGCGGGCCGTGCCCGACCGGATGTGCAGACACAATTACGA

GCTGGGAGGCCCCATGACCCTGCAGAGAAGAGTGCAGCCCAGAGTGAACGTGTCCCCCAGCAAGAAGGGCCCCC

TGCAGCACCACAACTTGCTTGTCTGCCACGTGACCGACTTCTACCCCGGCTCTATCCAAGTGCGGTGGTTCCTG

AACGGCCAGGAAGAGACAGCCGGCGTGGTGTCCACCAACCTGATCAGAAACGGCGACTGGACCTTCCAGATCCT

CGTGATGCTGGAAATGACCCCCCAGCAGGGCGACGTGTACACCTGTCAGGTGGAACACACCAGCCTGGACAGCC

CCGTGACCGTGGAATGGAAGGCCCAGAGCGATAGCGCCAGAAGCAAA
GGCGGCGGAGGCAGCCTGGAAATCGAG

GCCGCCTTCCTGGAAAGAGAGAACACCGCCCTGGAAACCCGGGTGGCCGAGCTGAGACAGAGAGTGCAGAGACT

GCGGAACCGGGTGTCCCAGTACCGGACCAGATATGGCCCTCTGGGAGGCGGCAAAGGGTCCGGCTTGAACGACA

TTTTTGAGGCCCAGAAGATAGAGTGGCACGAGTGA

Signal Peptide; DPB1*04: 01 custom-character

Extracellular Domain; Gly/Ser

Linker; Zip Sequences; GSlinker and biotinylation sequences) (SEQ ID NO:

258)

MMRPIVLVLLFATSALARATPENYLFQGRQECYAFNGTQRFLERYIYNREEFARFDSDVGEFRAVTELGRPAAE

YWNSQKDILEEKRAVPDRMCRHNYELGGPMTLQRRVQPRVNVSPSKKGPLQHHNWLVCHVTDFYPGSIQVRWFL

NGQEETAGVMSTNLIRNGDWTFQILVMLEMTPQQGDVYTCQVEHTSLDSPVTVEWKAQSDSARSKGGGGSLEIE

AAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGKGSGLNDIFEAQKIEWHE

Signal Peptide; DPB1*04: 01 custom-character

Extracellular Domain; Gly/Ser

Linker; Zip Sequences; GS linker and biotinylation sequences) (SEQ ID NO:

259)

ATGATGAGGCCCATCGTGCTGGTGCTGCTGTTCGCCACATCTGCCCTGGCC
AGAGCCACCCCCGAGAACTACCT

GTTTCAGGGCCGGCAGGAATGCTACGCCTTCAACGGCACCCAGCGGTTTCTGGAACGGTACATCTACAACCGGG

AAGAGTTCGCCAGATTCGACAGCGACGTGGGCGAGTTCAGAGCCGTGACAGAGCTGGGCAGACCTGCCGCCGAG

TACTGGAACAGCCAGAAGGACATCCTGGAAGAGAAGCGGGCCGTGCCCGACCGGATGTGCAGACACAATTACGA

GCTGGGAGGCCCCATGACCCTGCAGAGAAGAGTGCAGCCCAGAGTGAACGTGTCCCCCAGCAAGAAGGGCCCCC

TGCAGCACCACAAC
custom-character

CTTGTCTGCCACGTGACCGACTTCTACCCCGGCTCTATCCAAGTGCGGTGGTTCCTG

AACGGCCAGGAAGAGACAGCCGGCGTG
custom-character

TCCACCAACCTGATCAGAAACGGCGACTGGACCTTCCAGATCCT

CGTGATGCTGGAAATGACCCCCCAGCAGGGCGACGTGTACACCTGTCAGGTGGAACACACCAGCCTGGACAGCC

CCGTGACCGTGGAATGGAAGGCCCAGAGCGATAGCGCCAGAAGCAAA
GGCGGCGGAGGCAGCCTGGAAATCGAG

GCCGCCTTCCTGGAAAGAGAGAACACCGCCCTGGAAACCCGGGTGGCCGAGCTGAGACAGAGAGTGCAGAGACT

GCGGAACCGGGTGTCCCAGTACCGGACCAGATATGGCCCTCTGGGAGGCGGCAAAGGGTCCGGCTTGAACGACA

TTTTTGAGGCCCAGAAGATAGAGTGGCACGAGTGA

Alpha Chain

DPA1*01: 03 Extracellular Domain (SEQ ID NO: 6)

IKADHVSTYAAFVQTHRPTGEFMFEFDEDEMFYVDLDKKETVWHLEEFGQAFSFEAQGGLANIAILNNNLNTLI

QRSNHTQATNDPPEVTVFPKEPVELGQPNTLICHIDKFFPPVLNVTWLCNGELVTEGVAESLFLPRTDYSFHKF

HYLTFVPSAEDFYDCRVEHWGLDQPLLKHWEAQEPIQMPETTET

DPA1*01: 03 Extracellular Domain (SEQ ID NO: 7)

ATCAAGGCCGACCACGTGTCCACATACGCCGCCTTCGTGCAGACCCACAGACCCACCGGCGAGTTCATGTTCGA

GTTCGACGAGGACGAGATGTTCTACGTGGACCTGGACAAGAAAGAAACCGTGTGGCACCTGGAAGAGTTCGGCC

AGGCCTTCAGCTTTGAGGCCCAGGGCGGACTGGCCAATATCGCCATCCTGAACAACAACCTGAACACCCTGATC

CAGCGGAGCAACCACACCCAGGCCACCAACGATCCCCCCGAAGTGACCGTGTTCCCCAAAGAACCCGTGGAACT

GGGCCAGCCCAATACCCTGATCTGCCACATCGACAAGTTCTTCCCCCCCGTGCTGAACGTGACCTGGCTGTGCA

ATGGCGAGCTCGTGACAGAGGGCGTGGCCGAGTCTCTGTTCCTGCCCAGAACCGACTACAGCTTCCACAAGTTC

CACTACCTGACCTTCGTGCCCAGCGCCGAGGACTTCTACGACTGCAGAGTGGAACACTGGGGCCTGGACCAGCC

CCTGCTGAAACATTGGGAAGCCCAGGAACCCATCCAGATGCCCGAGACAACCGAGACA

Signal Peptide; DPA1*01: 03 Extracellular Domain; Gly/Ser Linker, Zip

Sequences and His tag sequences)(SEQ ID NO: 8)

MMRPIVLVLLFATSALAIKADHVSTYAAFVQTHRPTGEFMFEFDEDEMFYVDLDKKETVWHLEEFGQAFSFEAQ

GGLANIAILNNNLNTLIQRSNHTQATNDPPEVTVFPKEPVELGQPNTLICHIDKFFPPVLNVTWLCNGELVTEG

VAESLFLPRTDYSFHKFHYLTFVPSAEDFYDCRVEHWGLDQPLLKHWEAQEPIQMPETTETGGGGSLEIRAAFL

RQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGKGSHHHHHH

Signal Peptide; DPA1*01: 03 Extracellular Domain; Gly/Ser Linker, Zip

Sequences, and His tag sequences (10X) (SEQ ID NO: 260)

MMRPIVLVLLFATSALAIKADHVSTYAAFVQTHRPTGEFMFEFDEDEMFYVDLDKKETVWHLEEFGQAFSFEAQ

GGLANIAILNNNLNTLIQRSNHTQATNDPPEVTVFPKEPVELGQPNTLICHIDKFFPPVLNVTWLCNGELVTEG

VAESLFLPRTDYSFHKEHYLTFVPSAEDFYDCRVEHWGLDQPLLKHWEAQEPIQMPETTETGGGGSLEIRAAFL

RQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGKGSHHHHHHHHHH

Signal Peptide; DPA1*01: 03 Extracellular Domain; Gly/Ser Linker, Zip

Sequences, and His tag sequences (10X) (SEQ ID NO: 261)

ATGATGAGGCCCATCGTGCTGGTGCTGCTGTTCGCCACATCTGCCCTGGCCATCAAGGCCGACCACGTGTCCAC

ATACGCCGCCTTCGTGCAGACCCACAGACCCACCGGCGAGTTCATGTTCGAGTTCGACGAGGACGAGATGTTCT

ACGTGGACCTGGACAAGAAAGAAACCGTGTGGCACCTGGAAGAGTTCGGCCAGGCCTTCAGCTTTGAGGCCCAG

GGCGGACTGGCCAATATCGCCATCCTGAACAACAACCTGAACACCCTGATCCAGCGGAGCAACCACACCCAGGC

CACCAACGATCCCCCCGAAGTGACCGTGTTCCCCAAAGAACCCGTGGAACTGGGCCAGCCCAATACCCTGATCT

GCCACATCGACAAGTTCTTCCCCCCCGTGCTGAACGTGACCTGGCTGTGCAATGGCGAGCTCGTGACAGAGGGC

GTGGCCGAGTCTCTGTTCCTGCCCAGAACCGACTACAGCTTCCACAAGTTCCACTACCTGACCTTCGTGCCCAG

CGCCGAGGACTTCTACGACTGCAGAGTGGAACACTGGGGCCTGGACCAGCCCCTGCTGAAACATTGGGAAGCCC

AGGAACCCATCCAGATGCCCGAGACAACCGAGACAGGCGGCGGAGGCAGCCTGGAAATCAGAGCCGCCTTCCTG

CGGCAGAGAAACACCGCCCTGAGAACCGAAGTGGCCGAGCTGGAACAGGAAGTGCAGCGGCTGGAAAACGAGGT

GTCCCAGTACGAGACAAGATACGGCCCTCTGGGAGGCGGCAAGGGCTCTCACCACCACCATCACCATCATCATC

ACCATTGA

Signal Peptide (Fibroin light chain-derived)

MMRPIVLVLLFATSALA (SEQ ID NO: 9)

II.A.1.a. HLA-DP Beta Chain

In certain aspects, the HLA class II molecule comprises a DP beta chain, wherein the DP beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1. Any amino acid other than leucine can be present at the position corresponding to amino acid residue 112 of SEQ ID NO: 1. In some aspects, the amino acid other than leucine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is an amino acid selected from an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is an alanine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is a valine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is an isoleucine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is a methionine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is a phenylalanine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is a tyrosine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is a tryptophan.

In some embodiments, the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

In certain aspects, the HLA class II molecule comprises a DP beta chain, wherein the DP beta chain comprises an amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1. Any amino acid other than valine can be present at the position corresponding to amino acid residue 141 of SEQ ID NO: 1. In some aspects, the amino acid other than valine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO: 1 is an amino acid selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO: 1 is an alanine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO: 1 is an isoleucine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO: 1 is a leucine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO: 1 is a methionine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO: 1 is a phenylalanine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO: 1 is a tyrosine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO: 1 is a tryptophan.

In some aspects, the amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO: 1 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO: 1 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

In certain aspects of the present disclosure, the MHC class II molecule comprises a DP beta chain comprising more than one substitution mutation relative to the wild-type DP beta chain. In certain aspects, the DP beta chain comprises at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, at least eight mutations, at least nine mutations, or at least ten mutations relative to the wild-type DP beta chain.

In certain aspects, the DP beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1 and an amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1. In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1, (ii) the amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, or each of the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 and the amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1 is an amino acid comprising a hydrophobic side chain. In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is selected from an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan; and (ii) the amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1 is selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan.

In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is a tryptophan; and (ii) the amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1 is selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is selected from an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan; and (ii) the amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1 is a methionine. In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 is a tryptophan; and (ii) the amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1 is a methionine.

In certain aspects, the DP beta chain further comprises an amino acid other than valine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1. In some aspects, the amino acid other than valine at the position corresponding to amino acid residue 114 of SEQ ID NO: 1 is selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In certain aspects, the amino acid other than valine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1 is a methionine.

In certain aspects, the DP beta chain further comprises an amino acid other than methionine at a position corresponding to amino acid residue 158 of SEQ ID NO: 1. In some aspects, the amino acid other than methionine at the position corresponding to amino acid residue 158 of SEQ ID NO: 1 is selected from an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In certain aspects, the amino acid other than methionine at a position corresponding to amino acid residue 158 of SEQ ID NO: 1 is an isoleucine.

In some aspects, the DP beta chain comprises (i) an amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, and (ii) an amino acid other than valine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1. In some aspects, the DP beta chain comprises (i) an amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, and (ii) an amino acid other than methionine at a position corresponding to amino acid residue 158 of SEQ ID NO: 1.

In some aspects, the DP beta chain comprises (i) a tryptophan at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, and (ii) a methionine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1. In some aspects, the DP beta chain comprises (i) a tryptophan at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, and (ii) a isoleucine at a position corresponding to amino acid residue 158 of SEQ ID NO: 1.

In some aspects, the DP beta chain comprises (i) an amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, and (ii) an amino acid other than valine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1. In some aspects, the DP beta chain comprises (i) an amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, and (ii) an amino acid other than methionine at a position corresponding to amino acid residue 158 of SEQ ID NO: 1.

In some aspects, the DP beta chain comprises (i) a methionine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, and (ii) an amino acid other than valine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1. In some aspects, the DP beta chain comprises (i) a methionine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, and (ii) a isoleucine at a position corresponding to amino acid residue 158 of SEQ ID NO: 1.

In some aspects, the DP beta chain comprises (i) an amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, (ii) an amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, (iii) an amino acid other than valine at a position corresponding to amino acid residue 114 of SEQ ID NO: 1, and (iv) an amino acid other than methionine at a position corresponding to amino acid residue 158 of SEQ ID NO: 1.

In some aspects, the DP beta chain comprises a valine at a position corresponding to amino acid residue 114 SEQ ID NO: 1. In some aspects, the DP beta chain comprises a methionine at a position corresponding to amino acid residue 158 corresponding to SEQ ID NO: 1. In some aspects, the DP beta chain comprises (i) an amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, (ii) an amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, and (iii) a valine at a position corresponding to amino acid residue 114 SEQ ID NO: 1. In some aspects, the DP beta chain comprises (i) an amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, (ii) an amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, and (iii) a methionine at a position corresponding to amino acid residue 158 corresponding to SEQ ID NO: 1. In some aspects, the DP beta chain comprises (i) an amino acid other than leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, (ii) an amino acid other than valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, (iii) a valine at a position corresponding to amino acid residue 114 SEQ ID NO: 1, and (iv) a methionine at a position corresponding to amino acid residue 158 corresponding to SEQ ID NO: 1.

In some aspects, the DP beta chain comprises (i) a tryptophan at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, (ii) a methionine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, and (iii) a valine at a position corresponding to amino acid residue 114 SEQ ID NO: 1. In some aspects, the DP beta chain comprises (i) a tryptophan at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, (ii) a methionine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, and (iii) a methionine at a position corresponding to amino acid residue 158 corresponding to SEQ ID NO: 1. In some aspects, the DP beta chain comprises (i) a tryptophan at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, (ii) a methionine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, (iii) a valine at a position corresponding to amino acid residue 114 SEQ ID NO: 1, and (iv) a methionine at a position corresponding to amino acid residue 158 corresponding to SEQ ID NO: 1.

In certain aspects, a DP beta chain described herein has an increased affinity for a CD4 protein as compared to a reference HLA class II molecule. In some aspects, the reference HLA class II molecule is an HLA class II molecule having a wild-type DP beta chain. In some aspects, the reference HLA class II molecule is an HLA class II molecule having a DP beta chain comprising (i) a leucine at a position corresponding to amino acid residue 112 of SEQ ID NO: 1 and/or (ii) a valine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1.

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

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

In certain aspects, the DP beta chain comprises an allele selected from DPB1*01, DPB1*02, DPB1*03, DPB1*04, DPB1*05, DPB1*06, DPB1*08, DPB1*09, DPB1*10, DPB1*100, DPB1*101, DPB1*102, DPB1*103, DPB1*104, DPB1*105, DPB1*106, DPB1*107, DPB1*108, DPB1*109, DPB1*11, DPB1*110, DPB1*111, DPB1*112, DPB1*113, DPB1*114, DPB1*115, DPB1*116, DPB1*117, DPB1*118, DPB1*119, DPB1*120, DPB1*121, DPB1*122, DPB1*123, DPB1*124, DPB1*125, DPB1*126, DPB1*127, DPB1*128, DPB1*129, DPB1*13, DPB1*130, DPB1*131, DPB1*132, DPB1*133, DPB1*134, DPB1*135, DPB1*136, DPB1*137, DPB1*138, DPB1*139, DPB1*14, DPB1*140, DPB1*141, DPB1*142, DPB1*143, DPB1*144, DPB1*145, DPB1*146, DPB1*147, DPB1*148, DPB1*149, DPB1*15, DPB1*150, DPB1*151, DPB1*152, DPB1*153, DPB1*154, DPB1*155, DPB1*156, DPB1*157, DPB1*158, DPB1*159, DPB1*16, DPB1*160, DPB1*161, DPB1*162, DPB1*163, DPB1*164, DPB1*165, DPB1*166, DPB1*167, DPB1*168, DPB1*169, DPB1*17, DPB1*170, DPB1*171, DPB1*172, DPB1*173, DPB1*174, DPB1*175, DPB1*176, DPB1*177, DPB1*178, DPB1*179, DPB1*18, DPB1*180, DPB1*181, DPB1*182, DPB1*183, DPB1*184, DPB1*185, DPB1*186, DPB1*187, DPB1*188, DPB1*189, DPB1*19, DPB1*190, DPB1*191, DPB1*192, DPB1*193, DPB1*194, DPB1*195, DPB1*196, DPB1*197, DPB1*198, DPB1*199, DPB1*20, DPB1*200, DPB1*201, DPB1*202, DPB1*203, DPB1*204, DPB1*205, DPB1*206, DPB1*207, DPB1*208, DPB1*209, DPB1*21, DPB1*210, DPB1*211, DPB1*212, DPB1*213, DPB1*214, DPB1*215, DPB1*216, DPB1*217, DPB1*218, DPB1*219, DPB1*22, DPB1*220, DPB1*221, DPB1*222, DPB1*223, DPB1*224, DPB1*225, DPB1*226, DPB1*227, DPB1*228, DPB1*229, DPB1*23, DPB1*230, DPB1*231, DPB1*232, DPB1*233, DPB1*234, DPB1*235, DPB1*236, DPB1*237, DPB1*238, DPB1*239, DPB1*24, DPB1*240, DPB1*241, DPB1*242, DPB1*243, DPB1*244, DPB1*245, DPB1*246, DPB1*247, DPB1*248, DPB1*249, DPB1*25, DPB1*250, DPB1*251, DPB1*252, DPB1*253, DPB1*254, DPB1*255, DPB1*256, DPB1*257, DPB1*258, DPB1*259, DPB1*26, DPB1*260, DPB1*261, DPB1*262, DPB1*263, DPB1*264, DPB1*265, DPB1*266, DPB1*267, DPB1*268, DPB1*269, DPB1*27, DPB1*270, DPB1*271, DPB1*272, DPB1*273, DPB1*274, DPB1*275, DPB1*276, DPB1*277, DPB1*278, DPB1*279, DPB1*28, DPB1*280, DPB1*281, DPB1*282, DPB1*283, DPB1*284, DPB1*285, DPB1*286, DPB1*287, DPB1*288, DPB1*289, DPB1*29, DPB1*290, DPB1*291, DPB1*292, DPB1*293, DPB1*294, DPB1*295, DPB1*296, DPB1*297, DPB1*298, DPB1*299, DPB1*30, DPB1*300, DPB1*301, DPB1*302, DPB1*303, DPB1*304, DPB1*305, DPB1*306, DPB1*307, DPB1*308, DPB1*309, DPB1*31, DPB1*310, DPB1*311, DPB1*312, DPB1*313, DPB1*314, DPB1*315, DPB1*316, DPB1*317, DPB1*318, DPB1*319, DPB1*32, DPB1*320, DPB1*321, DPB1*322, DPB1*323, DPB1*324, DPB1*325, DPB1*326, DPB1*327, DPB1*328, DPB1*329, DPB1*33, DPB1*330, DPB1*331, DPB1*332, DPB1*333, DPB1*334, DPB1*335, DPB1*336, DPB1*337, DPB1*338, DPB1*339, DPB1*34, DPB1*340, DPB1*341, DPB1*342, DPB1*343, DPB1*344, DPB1*345, DPB1*346, DPB1*347, DPB1*348, DPB1*349, DPB1*35, DPB1*350, DPB1*351, DPB1*352, DPB1*353, DPB1*354, DPB1*355, DPB1*356, DPB1*357, DPB1*358, DPB1*359, DPB1*36, DPB1*360, DPB1*361, DPB1*362, DPB1*363, DPB1*364, DPB1*365, DPB1*366, DPB1*367, DPB1*368, DPB1*369, DPB1*37, DPB1*370, DPB1*371, DPB1*372, DPB1*373, DPB1*374, DPB1*375, DPB1*376, DPB1*377, DPB1*378, DPB1*379, DPB1*38, DPB1*380, DPB1*381, DPB1*382, DPB1*383, DPB1*384, DPB1*385, DPB1*386, DPB1*387, DPB1*388, DPB1*389, DPB1*39, DPB1*390, DPB1*391, DPB1*392, DPB1*393, DPB1*394, DPB1*395, DPB1*396, DPB1*397, DPB1*398, DPB1*399, DPB1*40, DPB1*400, DPB1*401, DPB1*402, DPB1*403, DPB1*404, DPB1*405, DPB1*406, DPB1*407, DPB1*408, DPB1*409, DPB1*41, DPB1*410, DPB1*411, DPB1*412, DPB1*413, DPB1*414, DPB1*415, DPB1*416, DPB1*417, DPB1*418, DPB1*419, DPB1*420, DPB1*421, DPB1*422, DPB1*423, DPB1*424, DPB1*425, DPB1*426, DPB1*427, DPB1*428, DPB1*429, DPB1*430, DPB1*431, DPB1*432, DPB1*433, DPB1*434, DPB1*435, DPB1*436, DPB1*437, DPB1*438, DPB1*439, DPB1*44, DPB1*440, DPB1*441, DPB1*442, DPB1*443, DPB1*444, DPB1*445, DPB1*446, DPB1*447, DPB1*448, DPB1*449, DPB1*45, DPB1*450, DPB1*451, DPB1*452, DPB1*453, DPB1*454, DPB1*455, DPB1*456, DPB1*457, DPB1*458, DPB1*459, DPB1*46, DPB1*460, DPB1*461, DPB1*462, DPB1*463, DPB1*464, DPB1*465, DPB1*466, DPB1*467, DPB1*468, DPB1*469, DPB1*47, DPB1*470, DPB1*471, DPB1*472, DPB1*473, DPB1*474, DPB1*475, DPB1*476, DPB1*477, DPB1*478, DPB1*479, DPB1*48, DPB1*480, DPB1*481, DPB1*482, DPB1*483, DPB1*484, DPB1*485, DPB1*486, DPB1*487, DPB1*488, DPB1*489, DPB1*49, DPB1*490, DPB1*491, DPB1*492, DPB1*493, DPB1*494, DPB1*495, DPB1*496, DPB1*497, DPB1*498, DPB1*499, DPB1*50, DPB1*500, DPB1*501, DPB1*502, DPB1*503, DPB1*504, DPB1*505, DPB1*506, DPB1*507, DPB1*508, DPB1*509, DPB1*51, DPB1*510, DPB1*511, DPB1*512, DPB1*513, DPB1*514, DPB1*515, DPB1*516, DPB1*517, DPB1*518, DPB1*519, DPB1*52, DPB1*520, DPB1*521, DPB1*522, DPB1*523, DPB1*524, DPB1*525, DPB1*526, DPB1*527, DPB1*528, DPB1*529, DPB1*53, DPB1*530, DPB1*531, DPB1*532, DPB1*533, DPB1*534, DPB1*535, DPB1*536, DPB1*537, DPB1*538, DPB1*539, DPB1*54, DPB1*540, DPB1*541, DPB1*542, DPB1*543, DPB1*544, DPB1*545, DPB1*546, DPB1*547, DPB1*548, DPB1*549, DPB1*55, DPB1*550, DPB1*551, DPB1*552, DPB1*553, DPB1*554, DPB1*555, DPB1*556, DPB1*557, DPB1*558, DPB1*559, DPB1*56, DPB1*560, DPB1*561, DPB1*562, DPB1*563, DPB1*564, DPB1*565, DPB1*566, DPB1*567, DPB1*568, DPB1*569, DPB1*57, DPB1*570, DPB1*571, DPB1*572, DPB1*573, DPB1*574, DPB1*575, DPB1*576, DPB1*577, DPB1*578, DPB1*579, DPB1*58, DPB1*580, DPB1*581, DPB1*582, DPB1*583, DPB1*584, DPB1*585, DPB1*586, DPB1*587, DPB1*588, DPB1*589, DPB1*59, DPB1*590, DPB1*591, DPB1*592, DPB1*593, DPB1*594, DPB1*595, DPB1*596, DPB1*597, DPB1*598, DPB1*599, DPB1*60, DPB1*600, DPB1*601, DPB1*602, DPB1*603, DPB1*604, DPB1*605, DPB1*606, DPB1*607, DPB1*608, DPB1*609, DPB1*61, DPB1*610, DPB1*611, DPB1*612, DPB1*613, DPB1*614, DPB1*615, DPB1*616, DPB1*617, DPB1*618, DPB1*619, DPB1*62, DPB1*620, DPB1*621, DPB1*622, DPB1*623, DPB1*624, DPB1*625, DPB1*626, DPB1*627, DPB1*628, DPB1*629, DPB1*63, DPB1*630, DPB1*631, DPB1*632, DPB1*633, DPB1*634, DPB1*635, DPB1*636, DPB1*637, DPB1*638, DPB1*639, DPB1*64, DPB1*640, DPB1*641, DPB1*642, DPB1*643, DPB1*644, DPB1*645, DPB1*646, DPB1*647, DPB1*648, DPB1*649, DPB1*65, DPB1*650, DPB1*651, DPB1*652, DPB1*653, DPB1*654, DPB1*655, DPB1*656, DPB1*657, DPB1*658, DPB1*659, DPB1*66, DPB1*660, DPB1*661, DPB1*662, DPB1*663, DPB1*664, DPB1*665, DPB1*666, DPB1*667, DPB1*668, DPB1*669, DPB1*67, DPB1*670, DPB1*671, DPB1*672, DPB1*673, DPB1*674, DPB1*675, DPB1*676, DPB1*677, DPB1*678, DPB1*679, DPB1*68, DPB1*680, DPB1*681, DPB1*682, DPB1*683, DPB1*684, DPB1*685, DPB1*686, DPB1*687, DPB1*688, DPB1*689, DPB1*69, DPB1*690, DPB1*691, DPB1*692, DPB1*693, DPB1*694, DPB1*695, DPB1*696, DPB1*697, DPB1*698, DPB1*699, DPB1*70, DPB1*700, DPB1*701, DPB1*702, DPB1*703, DPB1*704, DPB1*705, DPB1*706, DPB1*707, DPB1*708, DPB1*709, DPB1*71, DPB1*710, DPB1*711, DPB1*712, DPB1*713, DPB1*714, DPB1*715, DPB1*716, DPB1*717, DPB1*718, DPB1*719, DPB1*72, DPB1*720, DPB1*721, DPB1*722, DPB1*723, DPB1*724, DPB1*725, DPB1*726, DPB1*727, DPB1*728, DPB1*729, DPB1*73, DPB1*730, DPB1*731, DPB1*732, DPB1*733, DPB1*734, DPB1*735, DPB1*736, DPB1*737, DPB1*738, DPB1*739, DPB1*74, DPB1*740, DPB1*741, DPB1*742, DPB1*743, DPB1*744, DPB1*745, DPB1*746, DPB1*747, DPB1*748, DPB1*749, DPB1*75, DPB1*750, DPB1*751, DPB1*752, DPB1*753, DPB1*754, DPB1*755, DPB1*756, DPB1*757, DPB1*758, DPB1*759, DPB1*76, DPB1*760, DPB1*761, DPB1*762, DPB1*763, DPB1*764, DPB1*765, DPB1*766, DPB1*767, DPB1*768, DPB1*769, DPB1*77, DPB1*770, DPB1*771, DPB1*772, DPB1*773, DPB1*774, DPB1*775, DPB1*776, DPB1*777, DPB1*778, DPB1*779, DPB1*78, DPB1*780, DPB1*781, DPB1*782, DPB1*783, DPB1*784, DPB1*785, DPB1*786, DPB1*787, DPB1*788, DPB1*789, DPB1*79, DPB1*790, DPB1*791, DPB1*792, DPB1*794, DPB1*795, DPB1*796, DPB1*797, DPB1*798, DPB1*799, DPB1*80, DPB1*800, DPB1*801, DPB1*802, DPB1*803, DPB1*804, DPB1*805, DPB1*806, DPB1*807, DPB1*808, DPB1*809, DPB1*81, DPB1*810, DPB1*811, DPB1*812, DPB1*813, DPB1*814, DPB1*815, DPB1*816, DPB1*817, DPB1*818, DPB1*819, DPB1*82, DPB1*820, DPB1*821, DPB1*822, DPB1*823, DPB1*824, DPB1*825, DPB1*826, DPB1*827, DPB1*828, DPB1*829, DPB1*83, DPB1*830, DPB1*831, DPB1*832, DPB1*833, DPB1*834, DPB1*835, DPB1*836, DPB1*837, DPB1*838, DPB1*839, DPB1*84, DPB1*840, DPB1*841, DPB1*842, DPB1*843, DPB1*844, DPB1*845, DPB1*846, DPB1*847, DPB1*848, DPB1*849, DPB1*85, DPB1*850, DPB1*851, DPB1*852, DPB1*853, DPB1*854, DPB1*855, DPB1*856, DPB1*857, DPB1*858, DPB1*859, DPB1*86, DPB1*860, DPB1*861, DPB1*862, DPB1*863, DPB1*864, DPB1*865, DPB1*866, DPB1*867, DPB1*868, DPB1*869, DPB1*87, DPB1*870, DPB1*871, DPB1*872, DPB1*873, DPB1*874, DPB1*875, DPB1*876, DPB1*877, DPB1*878, DPB1*879, DPB1*88, DPB1*880, DPB1*881, DPB1*882, DPB1*883, DPB1*884, DPB1*885, DPB1*886, DPB1*887, DPB1*888, DPB1*889, DPB1*89, DPB1*890, DPB1*891, DPB1*892, DPB1*893, DPB1*894, DPB1*895, DPB1*896, DPB1*897, DPB1*898, DPB1*899, DPB1*90, DPB1*900, DPB1*901, DPB1*902, DPB1*903, DPB1*904, DPB1*905, DPB1*906, DPB1*907, DPB1*908, DPB1*909, DPB1*91, DPB1*910, DPB1*911, DPB1*912, DPB1*913, DPB1*914, DPB1*915, DPB1*916, DPB1*917, DPB1*918, DPB1*919, DPB1*92, DPB1*920, DPB1*921, DPB1*922, DPB1*923, DPB1*924, DPB1*925, DPB1*926, DPB1*927, DPB1*928, DPB1*929, DPB1*93, DPB1*930, DPB1*931, DPB1*932, DPB1*933, DPB1*934, DPB1*935, DPB1*936, DPB1*937, DPB1*938, DPB1*939, DPB1*94, DPB1*940, DPB1*941, DPB1*942, DPB1*943, DPB1*944, DPB1*945, DPB1*946, DPB1*947, DPB1*948, DPB1*949, DPB1*95, DPB1*950, DPB1*951, DPB1*952, DPB1*953, DPB1*954, DPB1*955, DPB1*956, DPB1*957, DPB1*958, DPB1*959, DPB1*96, DPB1*960, DPB1*961, DPB1*962, DPB1*963, DPB1*964, DPB1*965, DPB1*97, DPB1*98, and DPB1*99. In some aspects, the DP beta chain comprises an HLA-DPB1*01, HLA-DPB1*02, HLA-DPB1*03, HLA-DPB1*04, HLA-DPB1*05, HLA-DPB1*06, HLA-DPB1*08, or HLA-DPB1*09 allele. In certain aspects, the DP beta chain comprises an HLA-DPB1*04 allele. In particular aspects, the DP beta chain comprises an HLA-DPB1*04:01 allele.

In certain aspects, the MHC class II molecule comprises a DP beta chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 3, wherein the DP beta chain comprises a tryptophan at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, and wherein the DP beta chain comprises a methionine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1. In certain aspects, the MHC class II molecule comprises a DP beta chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 3, wherein the DP beta chain comprises (i) a tryptophan at a position corresponding to amino acid residue 112 of SEQ ID NO: 1, (ii) a methionine at a position corresponding to amino acid residue 141 of SEQ ID NO: 1, (iii) a valine at a position corresponding to amino acid residue 114 SEQ ID NO: 1, and (iv) a methionine at a position corresponding to amino acid residue 158 corresponding to SEQ ID NO: 1. In certain aspects, the MHC class II molecule comprises a DP beta chain comprising an amino acid sequence set forth in SEQ ID NO: 3.

II.A.1.a. HLA-DP Alpha Chain

In some aspects of the present disclosure, the MHC class II molecule further comprises an alpha chain. In some aspects, the alpha chain is a wild-type alpha chain. In some aspects, the alpha chain is a DP alpha chain. Any DP alpha chain can be used in the compositions and methods of the present disclosure. In some aspects, the DP alpha chain comprises an HLA-DPA1*01, HLA-DPA1*02, HLA-DPA1*03, or HLA-DPA1*04 allele. In certain aspects, the DP alpha chain comprises an HLA-DPA1*01 allele. In certain aspects, the DP alpha chain comprises an HLA-DPA1*02 allele. In certain aspects, the DP alpha chain comprises an HLA-DPA1*03 allele. In certain aspects, the DP alpha chain comprises an HLA-DPA1*04 allele.

In certain aspects, the DP alpha chain is selected from DPA1*01:03:01:01, DPA1*01:03:01:02, DPA1*01:03:01:03, DPA1*01:03:01:04, DPA1*01:03:01:05, DPA1*01:03:01:06, DPA1*01:03:01:07, DPA1*01:03:01:08, DPA1*01:03:01:09, DPA1*01:03:01:10, DPA1*01:03:01:11, DPA1*01:03:01:12, DPA1*01:03:01:13, DPA1*01:03:01:14, DPA1*01:03:01:15, DPA1*01:03:01:16, DPA1*01:03:01:17, DPA1*01:03:01:18Q, DPA1*01:03:01:19, DPA1*01:03:01:20, DPA1*01:03:01:21, DPA1*01:03:01:22, DPA1*01:03:01:23, DPA1*01:03:02, DPA1*01:03:03, DPA1*01:03:04, DPA1*01:03:05, DPA1*01:03:06, DPA1*01:03:07, DPA1*01:03:08, DPA1*01:03:09, DPA1*01:04, DPA1*01:05, DPA1*01:06:01, DPA1*01:06:02, DPA1*01:07, DPA1*01:08, DPA1*01:09, DPA1*01:10, DPA1*01:11, DPA1*01:12, DPA1*01:13, DPA1*01:14, DPA1*01:15, DPA1*01:16, DPA1*01:17, DPA1*01:18, DPA1*01:19, DPA1*02:01:01:01, DPA1*02:01:01:02, DPA1*02:01:01:03, DPA1*02:01:01:04, DPA1*02:01:01:05, DPA1*02:01:01:06, DPA1*02:01:01:07, DPA1*02:01:01:08, DPA1*02:01:01:09, DPA1*02:01:01:10, DPA1*02:01:01:11, DPA1*02:01:02:01, DPA1*02:01:02:02, DPA1*02:01:03, DPA1*02:01:04, DPA1*02:01:05, DPA1*02:01:06, DPA1*02:01:07, DPA1*02:01:08:01, DPA1*02:01:08:02, DPA1*02:02:02:01, DPA1*02:02:02:02, DPA1*02:02:02:03, DPA1*02:02:02:04, DPA1*02:02:02:05, DPA1*02:02:03, DPA1*02:02:04, DPA1*02:02:05, DPA1*02:02:06, DPA1*02:03, DPA1*02:04, DPA1*02:05, DPA1*02:06, DPA1*02:07:01:01, DPA1*02:07:01:02, DPA1*02:07:01:03, DPA1*02:08, DPA1*02:09, DPA1*02:10, DPA1*02:11, DPA1*02:12, DPA1*02:13N, DPA1*02:14, DPA1*02:15, DPA1*02:16, DPA1*03:01:01:01, DPA1*03:01:01:02, DPA1*03:01:01:03, DPA1*03:01:01:04, DPA1*03:01:01:05, DPA1*03:01:02, DPA1*03:02, DPA1*03:03, DPA1*03:04, DPA1*04:01:01:01, DPA1*04:01:01:02, DPA1*04:01:01:03, DPA1*04:02, or any combination thereof.

In certain aspects, the MHC class II molecule comprises a DP alpha chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 6. In certain aspects, the MHC class II molecule comprises a DP alpha chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 8. In certain aspects, the MHC class II molecule comprises a DP alpha chain comprising an amino acid sequence set forth in SEQ ID NO: 6. In certain aspects, the MHC class II molecule comprises a DP alpha chain comprising an amino acid sequence set forth in SEQ ID NO: 8.

II.A.2. HLA-DQ Molecules

Many HLA-DQ alleles are known in the art, and any of the known alleles can be used in the present disclosure. Examples of HLA-DQ alpha chain and beta chain alleles are shown in Table 4. An updated list of HLA alleles is available at hla.alleles.org/(last visited on Jul. 10, 2019).

TABLE 4

DQ Beta chain and alpha chain amino acid and nucleotide sequences.

Beta Chain

DQB1*05: 01 Extracellular Domain (SEQ ID NO: 11)

RDSPEDFVYQFKGLCYFTNGTERVRGVTRHIYNREEYVRFDSDVGVYRAVTPQGRPVAEYWNSQKEVLEGARAS

VDRVCRHNYEVAYRGILQRRVEPTVTISPSRTEALNHHNLLICSVTDFYPSQIKVRWFRNDQEETAGVVSTPLI

RNGDWTFQILVMLEMTPQRGDVYTCHVEHPSLQSPITVEWRAQSESAQSK

DQB1*05: 01 Extracellular Domain (SEQ ID NO: 12)

AGAGACTCTCCCGAGGATTTCGTGTACCAGTTTAAGGGCCTGTGCTACTTCACCAACGGGACGGAGCGCGTGCG

GGGTGTGACCAGACACATCTATAACCGAGAGGAGTACGTGCGCTTCGACAGCGACGTGGGGGTGTACCGGGCAG

TGACGCCGCAGGGGCGGCCTGTTGCCGAGTACTGGAACAGCCAGAAGGAAGTCCTGGAGGGGGCCCGGGCGTCG

GTGGACAGGGTGTGCAGACACAACTACGAGGTGGCGTACCGCGGGATCCTGCAGAGGAGAGTGGAGCCCACAGT

GACCATCTCCCCATCCAGGACAGAGGCCCTCAACCACCACAACCTGCTGATCTGCTCGGTGACAGATTTCTATC

CAAGCCAGATCAAAGTCCGGTGGTTTCGGAATGATCAGGAGGAGACAGCCGGCGTTGTGTCCACCCCCCTCATT

AGGAACGGTGACTGGACCTTCCAGATCCTGGTGATGCTGGAAATGACTCCCCAGCGTGGAGATGTCTACACCTG

CCACGTGGAGCACCCCAGCCTCCAGAGCCCCATCACCGTGGAGTGGCGGGCTCAGTCTGAATCTGCCCAGAGCA

AG

DQB1*05: 01 L114W/V143M Extracellular Domain (SEQ ID NO: 265)

RDSPEDFVYQFKGLCYFTNGTERVRGVTRHIYNREEYVRFDSDVGVYRAVTPQGRPVAEYWNSQKEVLEGARAS

VDRVCRHNYEVAYRGILQRRVEPTVTISPSRTEALNHHNWLICSVTDFYPSQIKVRWFRNDQEETAGVMSTPLI

RNGDWTFQILVMLEMTPQRGDVYTCHVEHPSLQSPITVEWRAQSESAQSK

DQB1*05: 01 L114W/V143M + 4 Reps Extracellular Domain (SEQ ID NO: 13)

RDSPEDEVYQFKGLCYFTNGTERVRGVTRHIYNREEYVRFDSDVGVYRAVTPQGRPVAEYWNSQKEVLEGARAS

VDRVCRHNYEVAYRGILQRRVEPTVTISPSRTEALQHHNWLVCHVTDFYPSQIKVRWFRNDQEETAGVMSTNLI

RNGDWTFQILVMLEMTPQRGDVYTCHVEHPSLQSPITVEWRAQSESAQSK

Signal Peptide; DQB1*05: 01 Domain; Gly/Ser Linker; Zip Sequences and His tag

sequences) (SEQ ID NO: 266)

MMRPIVLVLLFATSALARDSPEDFVYQFKGLCYFTNGTERVRGVTRHIYNREEYVRFDSDVGVYRAVTPQGRPV

AEYWNSQKEVLEGARASVDRVCRHNYEVAYRGILQRRVEPTVTISPSRTEALNHHNLLICSVTDFYPSQIKVRW

FRNDQEETAGVVSTPLIRNGDWTFQILVMLEMTPQRGDVYTCHVEHPSLQSPITVEWRAQSESAQSKGGGGSLE

IEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK

Signal Peptide; DQB1*05: 01 L114W/V143M Domain; Gly/Ser Linker; Zip Sequences

and His tag sequences) (SEQ ID NO: 267)

MMRPIVLVLLFATSALARDSPEDFVYQFKGLCYFTNGTERVRGVTRHIYNREEYVRFDSDVGVYRAVTPQGRPV

AEYWNSQKEVLEGARASVDRVCRHNYEVAYRGILQRRVEPTVTISPSRTEALNHHNWLICSVTDFYPSQIKVRW

FRNDQEETAGVMSTPLIRNGDWTFQILVMLEMTPQRGDVYTCHVEHPSLQSPITVEWRAQSESAQSKGGGGSLE

IEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK

Signal Peptide; DQB1*05: 01 L114W/V143M + 4 Reps Extracellular Domain; Gly/Ser

Linker; Zip Sequences and His tag sequences) (SEQ ID NO: 14)

MMRPIVLVLLFATSALA
RDSPEDFVYQFKGLCYFTNGTERVRGVTRHIYNREEYVRRDSDVGVYRAVTPQGRPV

AEYWNSQKEVLEGARASVDRVCRHNYEVAYRGILQRRVEPTVTISPSRTEALQHHNWLVCHVTDFYPSQIKVRW

FRNDQEETAGVMSTNLIRNGDWTFQILVMLEMTPQRGDVYTCHVEHPSLQSPITVEWRAQSESAQSK
GGGGSLE

IEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK

Full-length wild-type DQB1*05: 01 (SEQ ID NO: 15)

MSWKKSLRIPGDLRVATVTLMLAILSSSLAEGRDSPEDFVYQFKGLCYFTNGTERVRGVTRHIYNREEYVRFDS

DVGVYRAVTPQGRPVAEYWNSQKEVLEGARASVDRVCRHNYEVAYRGILQRRVEPTVTISPSRTEALNHHNLLI

CSVTDFYPSQIKVRWFRNDQEETAGVVSTPLIRNGDWTFQILVMLEMTPQRGDVYTCHVEHPSLQSPITVEWRA

QSESAQSKMLSGVGGFVLGLIFLGLGLIIRQRSRKGLLH

Alpha Chain

DQA1*01: 01 Extracellular Domain (SEQ ID NO: 16)

EDIVADHVASCGVNLYQFYGPSGQYTHEFDGDEEFYVDLERKETAWRWPEFSKFGGFDPQGALRNMAVAKHNLN

IMIKRYNSTAATNEVPEVTVFSKSPVTLGQPNTLICLVDNIFPPVVNITWLSNGQSVTEGVSETSFLSKSDHSF

FKISYLTFLPSADEIYDCKVEHWGLDQPLLKHWEPEIPAPMSELTET

DQA1*01: 01 Extracellular Domain (SEQ ID NO: 17)

GAGGACATCGTGGCCGATCACGTGGCAAGCTGCGGCGTGAACCTGTACCAGTTCTACGGCCCCTCTGGCCAGTA

CACCCATGAATTTGATGGAGATGAGGAGTTCTACGTGGACCTGGAGAGGAAGGAGACTGCCTGGCGGTGGCCTG

AGTTCAGCAAATTTGGAGGTTTTGACCCGCAGGGTGCACTGAGAAACATGGCTGTGGCAAAACACAACTTGAAC

ATCATGATTAAACGCTACAACTCTACCGCTGCTACCAATGAGGTTCCTGAGGTCACAGTGTTTTCCAAGTCTCC

CGTGACACTGGGTCAGCCCAACACCCTCATTTGTCTTGTGGACAACATCTTTCCTCCTGTGGTCAACATCACAT

GGCTGAGCAATGGGCAGTCAGTCACAGAAGGTGTTTCTGAGACCAGCTTCCTCTCCAAGAGTGATCATTCCTTC

TTCAAGATCAGTTACCTCACCTTCCTCCCTTCTGCTGATGAGATTTATGACTGCAAGGTGGAGCACTGGGGCCT

GGACCAGCCTCTTCTGAAACACTGGGAGCCTGAGATTCCAGCCCCTATGTCAGAGCTCACAGAGACT

Signal Peptide; DQA1*01: 01 Extracellular Domain; Gly/Ser Linker, Zip

Sequences and His tag sequences)(SEQ ID NO: 18)

MMRPIVLVLLFATSALAEDIVADHVASCGVNLYQFYGPSGQYTHEFDGDEEFYVDLERKETAWRWPEFSKEGGF

DPQGALRNMAVAKHNLNIMIKRYNSTAATNEVPEVTVFSKSPVTLGQPNTLICLVDNIFPPVVNITWLSNGQSV

TEGVSETSFLSKSDHSFFKISYLTFLPSADEIYDCKVEHWGLDQPLLKHWEPEIPAPMSELTETGGGGSLEIRA

AFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGKGSHHHHHH

II.A.2.a. HLA-DQ Beta Chain

In certain aspects, the HLA class II molecule comprises a DQ beta chain, wherein the DQ beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 11. Any amino acid other than leucine can be present at the position corresponding to amino acid residue 114 of SEQ ID NO: 11. In some aspects, the amino acid other than leucine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is an amino acid selected from an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is an alanine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is a valine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is an isoleucine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is a methionine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is a phenylalanine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is a tyrosine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is a tryptophan.

In some embodiments, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

In certain aspects, the HLA class II molecule comprises a DQ beta chain, wherein the DQ beta chain comprises an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11. Any amino acid other than valine can be present at the position corresponding to amino acid residue 143 of SEQ ID NO: 11. In some aspects, the amino acid other than valine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 11 is an amino acid selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 11 is an alanine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 11 is an isoleucine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 11 is a leucine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 11 is a methionine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 11 is a phenylalanine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 11 is a tyrosine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 11 is a tryptophan.

In some aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 11 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 11 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

In certain aspects, the HLA class II molecule comprises a DQ beta chain, wherein the DQ beta chain comprises an amino acid other than asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11. Any amino acid other than asparagine can be present at the position corresponding to amino acid residue 110 of SEQ ID NO: 11. In some aspects, the amino acid other than asparagine is an amino acid comprising a polar uncharged side chain. In certain aspects, the amino acid other than asparagine at the position corresponding to amino acid residue 110 of SEQ ID NO: 11 is an amino acid selected from a serine, a threonine, and a glutamine. In certain aspects, the amino acid other than asparagine at the position corresponding to amino acid residue 110 of SEQ ID NO: 11 is a serine. In certain aspects, the amino acid other than asparagine at the position corresponding to amino acid residue 110 of SEQ ID NO: 11 is a threonine. In certain aspects, the amino acid other than asparagine at the position corresponding to amino acid residue 110 of SEQ ID NO: 11 is a glutamine.

In some aspects, the amino acid other than asparagine at the position corresponding to amino acid residue 110 of SEQ ID NO: 11 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a polar uncharged side chain. In certain aspects, the amino acid other than asparagine at the position corresponding to amino acid residue 110 of SEQ ID NO: 11 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a polar uncharged side chain.

In certain aspects, the HLA class II molecule comprises a DQ beta chain, wherein the DQ beta chain comprises an amino acid other than isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11. Any amino acid other than isoleucine can be present at the position corresponding to amino acid residue 116 of SEQ ID NO: 11. In some aspects, the amino acid other than isoleucine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than isoleucine at the position corresponding to amino acid residue 116 of SEQ ID NO: 11 is an amino acid selected from an alanine, a valine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In certain aspects, the amino acid other than isoleucine at the position corresponding to amino acid residue 116 of SEQ ID NO: 11 is an alanine. In certain aspects, the amino acid other than isoleucine at the position corresponding to amino acid residue 116 of SEQ ID NO: 11 is a valine. In certain aspects, the amino acid other than isoleucine at the position corresponding to amino acid residue 116 of SEQ ID NO: 11 is a leucine. In certain aspects, the amino acid other than isoleucine at the position corresponding to amino acid residue 116 of SEQ ID NO: 11 is a methionine. In certain aspects, the amino acid other than isoleucine at the position corresponding to amino acid residue 116 of SEQ ID NO: 11 is a phenylalanine. In certain aspects, the amino acid other than isoleucine at the position corresponding to amino acid residue 116 of SEQ ID NO: 11 is a tyrosine. In certain aspects, the amino acid other than isoleucine at the position corresponding to amino acid residue 116 of SEQ ID NO: 11 is a tryptophan.

In some aspects, the amino acid other than isoleucine at the position corresponding to amino acid residue 116 of SEQ ID NO: 11 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than isoleucine at the position corresponding to amino acid residue 116 of SEQ ID NO: 11 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

In certain aspects, the HLA class II molecule comprises a DQ beta chain, wherein the DQ beta chain comprises an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11. Any amino acid other than serine can be present at the position corresponding to amino acid residue 118 of SEQ ID NO: 11. In some aspects, the amino acid other than serine is an amino acid comprising an electrically charged side chain. In certain aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 11 is an amino acid selected from an arginine, a histidine, and a lysine. In certain aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 11 is an arginine. In certain aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 11 is a histidine. In certain aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 11 is a lysine.

In some aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 11 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises an electrically charged side chain. In certain aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 11 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises an electrically charged side chain.

In certain aspects, the HLA class II molecule comprises a DQ beta chain, wherein the DQ beta chain comprises an amino acid other than proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11. Any amino acid other than proline can be present at the position corresponding to amino acid residue 146 of SEQ ID NO: 11. In some aspects, the amino acid other than proline is an amino acid comprising a polar uncharged side chain. In certain aspects, the amino acid other than proline at the position corresponding to amino acid residue 146 of SEQ ID NO: 11 is an amino acid selected from a serine, a threonine, an asparagine, and a glutamine. In certain aspects, the amino acid other than proline at the position corresponding to amino acid residue 146 of SEQ ID NO: 11 is a serine. In certain aspects, the amino acid other than proline at the position corresponding to amino acid residue 146 of SEQ ID NO: 11 is a threonine. In certain aspects, the amino acid other than proline at the position corresponding to amino acid residue 146 of SEQ ID NO: 11 is an asparagine. In certain aspects, the amino acid other than proline at the position corresponding to amino acid residue 146 of SEQ ID NO: 11 is a glutamine.

In some aspects, the amino acid other than proline at the position corresponding to amino acid residue 146 of SEQ ID NO: 11 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a polar uncharged side chain. In certain aspects, the amino acid other than proline at the position corresponding to amino acid residue 146 of SEQ ID NO: 11 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a polar uncharged side chain.

In certain aspects of the present disclosure, the MHC class II molecule comprises a DQ beta chain comprising more than one substitution mutation relative to the wild-type DQ beta chain. In certain aspects, the DQ beta chain comprises at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, at least eight mutations, at least nine mutations, or at least ten mutations relative to the wild-type DQ beta chain.

In certain aspects, the DQ beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 11 and an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11. In certain aspects, the DQ beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 11; an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11; and at least three of: (i) an amino acid other than asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11, (ii) an amino acid other than isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11, (iii) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11, and (iv) an amino acid other than proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11.

In some aspects, the DQ beta chain comprises (i) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 11; (ii) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11; (iii) an amino acid other than asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11; (iv) an amino acid other than isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11; (v) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11; and (vi) an amino acid other than proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11.

In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11, (ii) the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11, or each of the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 and the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11 is an amino acid comprising a hydrophobic side chain.

In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is selected from an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan; (ii) the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11 is selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan; (iii) the amino acid other than asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11 is selected from a serine, a threonine, and a glutamine; (iv) the amino acid other than isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11 is selected from an alanine, a valine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan; (v) the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11 is selected from an arginine, a histidine, and a lysine; and (vi) the amino acid other than proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11 is selected from a serine, a threonine, an asparagine, and a glutamine.

In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is a tryptophan; (ii) the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11 is selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is selected from an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan; and (ii) the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11 is a methionine. In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is a tryptophan; and (ii) the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11 is a methionine. In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 11 is a tryptophan; (ii) the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11 is a methionine; (iii) the amino acid other than asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11 is a glutamine; (iv) the amino acid other than isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11 is a valine; (v) the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11 is a histidine; and (vi) the amino acid other than proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11 is a glutamine.

In certain aspects, a DQ beta chain described herein has an increased affinity for a CD4 protein as compared to a reference HLA class II molecule. In some aspects, the reference HLA class II molecule is an HLA class II molecule having a wild-type DQ beta chain. In some aspects, the reference HLA class II molecule is an HLA class II molecule having a DQ beta chain comprising (i) a leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 11 and/or (ii) a valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11. In some aspects, the reference HLA class II molecule is an HLA class II molecule having a DQ beta chain comprising (i) a leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 11, (ii) a valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11, (iii) an asparagine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11, (iv) an isoleucine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11, (iii) a serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11, and/or (iv) a proline at a position corresponding to amino acid residue 146 of SEQ ID NO: 11.

In certain aspects, the DQ beta chain comprises an allele selected from an HLA-DQB1*02, an HLA-DQB1*03, an HLA-DQB1*04, an HLA-DQB1*05, and an HLA-DQB1*06 allele. In certain aspects, the DQ beta chain comprises an HLA-DQB1*05 allele. In particular aspects, the DQ beta chain comprises an HLA-DQB1*05:01 allele.

In certain aspects, the DQ beta chain comprises an allele selected from DQB1*02:01:01, DQB1*02:01:02, DQB1*02:01:03, DQB1*02:01:04, DQB1*02:01:05, DQB1*02:01:06, DQB1*02:01:07, DQB1*02:01:08, DQB1*02:01:09, DQB1*02:01:10, DQB1*02:01:11, DQB1*02:01:12, DQB1*02:01:13, DQB1*02:01:14, DQB1*02:01:15, DQB1*02:01:16, DQB1*02:01:17, DQB1*02:01:18, DQB1*02:01:19, DQB1*02:01:20, DQB1*02:01:21, DQB1*02:01:22, DQB1*02:01:23, DQB1*02:01:24, DQB1*02:01:25, DQB1*02:01:26, DQB1*02:01:27, DQB1*02:01:28, DQB1*02:01:29, DQB1*02:01:30, DQB1*02:01:31, DQB1*02:02:01:01, DQB1*02:02:01:02, DQB1*02:02:01:03, DQB1*02:02:01:04, DQB1*02:02:02, DQB1*02:02:03, DQB1*02:02:04, DQB1*02:02:05, DQB1*02:02:06, DQB1*02:02:07, DQB1*02:02:08, DQB1*02:02:09, DQB1*02:03:01, DQB1*02:03:02, DQB1*02:04, DQB1*02:05, DQB1*02:06, DQB1*02:07:01, DQB1*02:07:02, DQB1*02:08, DQB1*02:09, DQB1*02:10, DQB1*02:100, DQB1*02:101, DQB1*02:102, DQB1*02:103, DQB1*02:104, DQB1*02:105, DQB1*02:106, DQB1*02:107, DQB1*02:108, DQB1*02:109, DQB1*02:11, DQB1*02:110, DQB1*02:111, DQB1*02:112, DQB1*02:113, DQB1*02:114, DQB1*02:115, DQB1*02:116, DQB1*02:117, DQB1*02:118, DQB1*02:119, DQB1*02:12, DQB1*02:120, DQB1*02:121, DQB1*02:122, DQB1*02:123, DQB1*02:124, DQB1*02:125, DQB1*02:126, DQB1*02:127, DQB1*02:128, DQB1*02:129N, DQB1*02:13, DQB1*02:130, DQB1*02:131, DQB1*02:132N, DQB1*02:133, DQB1*02:134N, DQB1*02:135, DQB1*02:136, DQB1*02:137, DQB1*02:138, DQB1*02:139, DQB1*02:140, DQB1*02:141, DQB1*02:142, DQB1*02:14:01, DQB1*02:14:02, DQB1*02:15, DQB1*02:16, DQB1*02:17, DQB1*02:18N, DQB1*02:19, DQB1*02:20N, DQB1*02:21, DQB1*02:22, DQB1*02:23, DQB1*02:24, DQB1*02:25, DQB1*02:26, DQB1*02:27, DQB1*02:28, DQB1*02:29, DQB1*02:30, DQB1*02:31, DQB1*02:32, DQB1*02:33, DQB1*02:34, DQB1*02:35, DQB1*02:36, DQB1*02:37, DQB1*02:38, DQB1*02:39, DQB1*02:40, DQB1*02:41, DQB1*02:42, DQB1*02:43, DQB1*02:44, DQB1*02:45, DQB1*02:46, DQB1*02:47, DQB1*02:48, DQB1*02:49, DQB1*02:50, DQB1*02:51, DQB1*02:52, DQB1*02:53Q, DQB1*02:54, DQB1*02:55, DQB1*02:56, DQB1*02:57, DQB1*02:58N, DQB1*02:59, DQB1*02:60, DQB1*02:61, DQB1*02:62, DQB1*02:63, DQB1*02:64, DQB1*02:65, DQB1*02:66, DQB1*02:67NX, DQB1*02:68, DQB1*02:69, DQB1*02:70, DQB1*02:71, DQB1*02:72, DQB1*02:73, DQB1*02:74, DQB1*02:75, DQB1*02:76, DQB1*02:77, DQB1*02:78, DQB1*02:79, DQB1*02:80, DQB1*02:81, DQB1*02:82, DQB1*02:83, DQB1*02:84, DQB1*02:85, DQB1*02:86, DQB1*02:87, DQB1*02:88, DQB1*02:89:01, DQB1*02:89:02, DQB1*02:90, DQB1*02:91, DQB1*02:92, DQB1*02:93, DQB1*02:94, DQB1*02:95, DQB1*02:96N, DQB1*02:97, DQB1*02:98, DQB1*02:99, DQB1*03:01:01:01, DQB1*03:01:01:02, DQB1*03:01:01:03, DQB1*03:01:01:04, DQB1*03:01:01:05, DQB1*03:01:01:06, DQB1*03:01:01:07, DQB1*03:01:01:08, DQB1*03:01:01:09, DQB1*03:01:01:10, DQB1*03:01:01:11, DQB1*03:01:01:12, DQB1*03:01:01:14, DQB1*03:01:01:15, DQB1*03:01:01:16, DQB1*03:01:01:17, DQB1*03:01:01:18, DQB1*03:01:01:19, DQB1*03:01:01:20, DQB1*03:01:02, DQB1*03:01:03, DQB1*03:01:04, DQB1*03:01:05, DQB1*03:01:06, DQB1*03:01:07, DQB1*03:01:08, DQB1*03:01:09, DQB1*03:01:10, DQB1*03:01:11, DQB1*03:01:12, DQB1*03:01:13, DQB1*03:01:14, DQB1*03:01:15, DQB1*03:01:16, DQB1*03:01:17, DQB1*03:01:18, DQB1*03:01:19, DQB1*03:01:20, DQB1*03:01:21, DQB1*03:01:22, DQB1*03:01:23, DQB1*03:01:24, DQB1*03:01:25, DQB1*03:01:26, DQB1*03:01:27, DQB1*03:01:28, DQB1*03:01:29, DQB1*03:01:30, DQB1*03:01:31, DQB1*03:01:32, DQB1*03:01:33, DQB1*03:01:34, DQB1*03:01:35, DQB1*03:01:36, DQB1*03:01:37, DQB1*03:01:38, DQB1*03:01:39, DQB1*03:01:40, DQB1*03:01:41, DQB1*03:01:42, DQB1*03:01:43, DQB1*03:01:44, DQB1*03:01:45, DQB1*03:01:46, DQB1*03:02:01:01, DQB1*03:02:01:02, DQB1*03:02:01:03, DQB1*03:02:01:04, DQB1*03:02:01:05, DQB1*03:02:01:06, DQB1*03:02:01:07, DQB1*03:02:01:08, DQB1*03:02:02, DQB1*03:02:03, DQB1*03:02:04, DQB1*03:02:05, DQB1*03:02:06, DQB1*03:02:07, DQB1*03:02:08, DQB1*03:02:09, DQB1*03:02:10, DQB1*03:02:11, DQB1*03:02:12, DQB1*03:02:13, DQB1*03:02:14, DQB1*03:02:15, DQB1*03:02:16, DQB1*03:02:17, DQB1*03:02:18, DQB1*03:02:19, DQB1*03:02:20, DQB1*03:02:21, DQB1*03:02:22, DQB1*03:02:23, DQB1*03:02:24, DQB1*03:02:25, DQB1*03:02:26, DQB1*03:02:27, DQB1*03:02:28, DQB1*03:02:29, DQB1*03:02:30, DQB1*03:03:02:01, DQB1*03:03:02:02, DQB1*03:03:02:03, DQB1*03:03:02:04, DQB1*03:03:02:05, DQB1*03:03:03, DQB1*03:03:04, DQB1*03:03:05, DQB1*03:03:06, DQB1*03:03:07, DQB1*03:03:08, DQB1*03:03:09, DQB1*03:03:10, DQB1*03:03:11, DQB1*03:03:12, DQB1*03:03:13, DQB1*03:03:14, DQB1*03:03:15, DQB1*03:03:16, DQB1*03:03:17, DQB1*03:03:18, DQB1*03:03:19, DQB1*03:03:20, DQB1*03:03:21, DQB1*03:04:01, DQB1*03:04:02, DQB1*03:04:03, DQB1*03:04:04, DQB1*03:05:01, DQB1*03:05:02, DQB1*03:05:03, DQB1*03:05:04, DQB1*03:06, DQB1*03:07, DQB1*03:08, DQB1*03:09, DQB1*03:100, DQB1*03:101, DQB1*03:102, DQB1*03:103, DQB1*03:104, DQB1*03:105, DQB1*03:106, DQB1*03:107, DQB1*03:108, DQB1*03:109, DQB1*03:10:01, DQB1*03:10:02:01, DQB1*03:10:02:02, DQB1*03:11, DQB1*03:110, DQB1*03:111, DQB1*03:112, DQB1*03:113, DQB1*03:114, DQB1*03:115, DQB1*03:116, DQB1*03:117, DQB1*03:118N, DQB1*03:119, DQB1*03:12, DQB1*03:120, DQB1*03:121, DQB1*03:122, DQB1*03:123, DQB1*03:124, DQB1*03:125, DQB1*03:126, DQB1*03:127, DQB1*03:128, DQB1*03:129, DQB1*03:13, DQB1*03:130, DQB1*03:131, DQB1*03:132, DQB1*03:133, DQB1*03:134, DQB1*03:135, DQB1*03:136, DQB1*03:137, DQB1*03:138, DQB1*03:139, DQB1*03:140, DQB1*03:141, DQB1*03:142, DQB1*03:143, DQB1*03:144, DQB1*03:145, DQB1*03:146, DQB1*03:147, DQB1*03:148, DQB1*03:149, DQB1*03:14:01, DQB1*03:14:02, DQB1*03:15, DQB1*03:150, DQB1*03:151, DQB1*03:152, DQB1*03:153, DQB1*03:154, DQB1*03:155, DQB1*03:156, DQB1*03:157, DQB1*03:158, DQB1*03:159, DQB1*03:16, DQB1*03:160, DQB1*03:161, DQB1*03:162, DQB1*03:163, DQB1*03:164, DQB1*03:165, DQB1*03:166, DQB1*03:167, DQB1*03:168, DQB1*03:169, DQB1*03:170, DQB1*03:171, DQB1*03:172, DQB1*03:173, DQB1*03:174, DQB1*03:175, DQB1*03:176, DQB1*03:177, DQB1*03:178, DQB1*03:179, DQB1*03:17:01, DQB1*03:17:02, DQB1*03:18, DQB1*03:180, DQB1*03:181, DQB1*03:182, DQB1*03:183, DQB1*03:184, DQB1*03:185, DQB1*03:186, DQB1*03:187, DQB1*03:188, DQB1*03:189, DQB1*03:190, DQB1*03:191, DQB1*03:192, DQB1*03:193, DQB1*03:194, DQB1*03:195, DQB1*03:196, DQB1*03:197Q, DQB1*03:198:01, DQB1*03:198:02, DQB1*03:199, DQB1*03:19:01, DQB1*03:19:02, DQB1*03:19:03, DQB1*03:19:04, DQB1*03:20, DQB1*03:200, DQB1*03:201, DQB1*03:202, DQB1*03:203, DQB1*03:204, DQB1*03:205, DQB1*03:206, DQB1*03:207, DQB1*03:208, DQB1*03:209, DQB1*03:21, DQB1*03:210, DQB1*03:211, DQB1*03:212, DQB1*03:213NX, DQB1*03:214, DQB1*03:215, DQB1*03:216, DQB1*03:217, DQB1*03:218, DQB1*03:219, DQB1*03:220, DQB1*03:221, DQB1*03:222, DQB1*03:223, DQB1*03:224, DQB1*03:225, DQB1*03:226, DQB1*03:227, DQB1*03:228, DQB1*03:229, DQB1*03:22:01, DQB1*03:22:02, DQB1*03:230, DQB1*03:231, DQB1*03:232, DQB1*03:233, DQB1*03:234, DQB1*03:235, DQB1*03:236, DQB1*03:237N, DQB1*03:238, DQB1*03:239, DQB1*03:23:01, DQB1*03:23:02, DQB1*03:23:03, DQB1*03:24, DQB1*03:240, DQB1*03:241, DQB1*03:242, DQB1*03:243, DQB1*03:244, DQB1*03:245, DQB1*03:246, DQB1*03:247, DQB1*03:248, DQB1*03:249, DQB1*03:250, DQB1*03:251, DQB1*03:252, DQB1*03:253, DQB1*03:254, DQB1*03:255, DQB1*03:256, DQB1*03:257, DQB1*03:258, DQB1*03:259, DQB1*03:25:01, DQB1*03:25:02, DQB1*03:26, DQB1*03:260, DQB1*03:261, DQB1*03:262, DQB1*03:263, DQB1*03:264, DQB1*03:265, DQB1*03:266, DQB1*03:267, DQB1*03:268, DQB1*03:269N, DQB1*03:27, DQB1*03:270, DQB1*03:271, DQB1*03:272, DQB1*03:273, DQB1*03:274, DQB1*03:275, DQB1*03:277, DQB1*03:278, DQB1*03:279, DQB1*03:28, DQB1*03:280, DQB1*03:281, DQB1*03:282N, DQB1*03:283, DQB1*03:284, DQB1*03:285, DQB1*03:286, DQB1*03:287, DQB1*03:288, DQB1*03:289, DQB1*03:29, DQB1*03:290, DQB1*03:291, DQB1*03:292, DQB1*03:293, DQB1*03:294, DQB1*03:295, DQB1*03:296, DQB1*03:297, DQB1*03:298, DQB1*03:299, DQB1*03:30, DQB1*03:300, DQB1*03:301, DQB1*03:302, DQB1*03:303N, DQB1*03:304, DQB1*03:305, DQB1*03:306, DQB1*03:307, DQB1*03:308, DQB1*03:309, DQB1*03:31, DQB1*03:310N, DQB1*03:311, DQB1*03:312, DQB1*03:313, DQB1*03:314, DQB1*03:315, DQB1*03:316, DQB1*03:317:01, DQB1*03:317:02, DQB1*03:318, DQB1*03:319, DQB1*03:32, DQB1*03:320, DQB1*03:321, DQB1*03:322, DQB1*03:323, DQB1*03:324, DQB1*03:326, DQB1*03:327, DQB1*03:328, DQB1*03:329, DQB1*03:33, DQB1*03:330, DQB1*03:331, DQB1*03:332, DQB1*03:333, DQB1*03:334N4 bp, DQB1*03:335, DQB1*03:336, DQB1*03:337, DQB1*03:338N, DQB1*03:339N, DQB1*03:34, DQB1*03:340N, DQB1*03:341, DQB1*03:342, DQB1*03:343, DQB1*03:344, DQB1*03:345, DQB1*03:346, DQB1*03:347, DQB1*03:348, DQB1*03:349, DQB1*03:35, DQB1*03:350, DQB1*03:351, DQB1*03:352, DQB1*03:353, DQB1*03:354N, DQB1*03:355, DQB1*03:356NX, DQB1*03:357N, DQB1*03:358N, DQB1*03:36, DQB1*03:37, DQB1*03:38:01, DQB1*03:38:02, DQB1*03:39, DQB1*03:40, DQB1*03:41, DQB1*03:42, DQB1*03:43, DQB1*03:44, DQB1*03:45, DQB1*03:46, DQB1*03:47, DQB1*03:48, DQB1*03:49, DQB1*03:50, DQB1*03:51, DQB1*03:52, DQB1*03:53, DQB1*03:54, DQB1*03:55, DQB1*03:56, DQB1*03:57, DQB1*03:58, DQB1*03:59, DQB1*03:60, DQB1*03:61, DQB1*03:62, DQB1*03:63, DQB1*03:64, DQB1*03:65, DQB1*03:66N, DQB1*03:67, DQB1*03:68, DQB1*03:69, DQB1*03:70, DQB1*03:71, DQB1*03:72, DQB1*03:73, DQB1*03:74, DQB1*03:75, DQB1*03:76, DQB1*03:77, DQB1*03:78, DQB1*03:79, DQB1*03:80, DQB1*03:81, DQB1*03:82, DQB1*03:83, DQB1*03:84N, DQB1*03:85, DQB1*03:86, DQB1*03:87, DQB1*03:88, DQB1*03:89, DQB1*03:90N, DQB1*03:91Q, DQB1*03:92, DQB1*03:93, DQB1*03:94, DQB1*03:95N, DQB1*03:96, DQB1*03:97, DQB1*03:98, DQB1*03:99Q, DQB1*04:01:01:01, DQB1*04:01:01:02, DQB1*04:01:02, DQB1*04:01:03, DQB1*04:01:04, DQB1*04:01:05, DQB1*04:02:01:01, DQB1*04:02:01:04, DQB1*04:02:01:05, DQB1*04:02:01:06, DQB1*04:02:01:07, DQB1*04:02:01:08, DQB1*04:02:01:09, DQB1*04:02:01:10, DQB1*04:02:02, DQB1*04:02:03, DQB1*04:02:04, DQB1*04:02:05, DQB1*04:02:06, DQB1*04:02:07, DQB1*04:02:08, DQB1*04:02:09, DQB1*04:02:10, DQB1*04:02:11, DQB1*04:02:12, DQB1*04:02:13, DQB1*04:02:14, DQB1*04:02:15, DQB1*04:02:16, DQB1*04:02:17, DQB1*04:02:18, DQB1*04:03:01, DQB1*04:03:02, DQB1*04:03:03, DQB1*04:04, DQB1*04:05, DQB1*04:06, DQB1*04:07, DQB1*04:08, DQB1*04:09, DQB1*04:10, DQB1*04:11, DQB1*04:12, DQB1*04:13, DQB1*04:14, DQB1*04:15, DQB1*04:16, DQB1*04:17, DQB1*04:18, DQB1*04:19, DQB1*04:20, DQB1*04:21, DQB1*04:22, DQB1*04:23, DQB1*04:24, DQB1*04:25N, DQB1*04:26, DQB1*04:27, DQB1*04:28, DQB1*04:29, DQB1*04:30, DQB1*04:31, DQB1*04:32, DQB1*04:33, DQB1*04:34, DQB1*04:35, DQB1*04:36N, DQB1*04:37, DQB1*04:38, DQB1*04:39, DQB1*04:40, DQB1*04:41N, DQB1*04:42, DQB1*04:43, DQB1*04:44, DQB1*04:45, DQB1*04:46N, DQB1*04:47, DQB1*04:48, DQB1*04:49, DQB1*04:50, DQB1*04:51, DQB1*04:52, DQB1*04:53, DQB1*04:54, DQB1*04:55, DQB1*04:56, DQB1*04:57, DQB1*04:58, DQB1*04:59N, DQB1*04:60, DQB1*04:61, DQB1*04:62, DQB1*05:01:01:01, DQB1*05:01:01:02, DQB1*05:01:01:03, DQB1*05:01:01:04, DQB1*05:01:01:05, DQB1*05:01:02, DQB1*05:01:03, DQB1*05:01:04, DQB1*05:01:05, DQB1*05:01:06, DQB1*05:01:07, DQB1*05:01:08, DQB1*05:01:09, DQB1*05:01:10, DQB1*05:01:11, DQB1*05:01:12, DQB1*05:01:13, DQB1*05:01:14, DQB1*05:01:15, DQB1*05:01:16, DQB1*05:01:17, DQB1*05:01:18, DQB1*05:01:19, DQB1*05:01:20, DQB1*05:01:21, DQB1*05:01:22, DQB1*05:01:23, DQB1*05:01:24:01, DQB1*05:01:24:02, DQB1*05:01:25, DQB1*05:01:26, DQB1*05:01:27, DQB1*05:01:28, DQB1*05:01:29, DQB1*05:01:30, DQB1*05:01:31, DQB1*05:01:32, DQB1*05:01:33, DQB1*05:01:34, DQB1*05:02:01:01, DQB1*05:02:01:02, DQB1*05:02:01:03, DQB1*05:02:01:04, DQB1*05:02:01:05, DQB1*05:02:01:06, DQB1*05:02:02, DQB1*05:02:03, DQB1*05:02:04, DQB1*05:02:05, DQB1*05:02:06, DQB1*05:02:07, DQB1*05:02:08, DQB1*05:02:09, DQB1*05:02:10, DQB1*05:02:11, DQB1*05:02:12, DQB1*05:02:13, DQB1*05:02:14, DQB1*05:02:15, DQB1*05:02:16, DQB1*05:02:17, DQB1*05:02:18, DQB1*05:02:19, DQB1*05:03:01:01, DQB1*05:03:01:02, DQB1*05:03:01:03, DQB1*05:03:02, DQB1*05:03:03, DQB1*05:03:04, DQB1*05:03:05, DQB1*05:03:06, DQB1*05:03:07, DQB1*05:03:08, DQB1*05:03:09, DQB1*05:03:10, DQB1*05:03:11, DQB1*05:03:12, DQB1*05:03:13, DQB1*05:03:14, DQB1*05:03:15, DQB1*05:03:16, DQB1*05:03:17, DQB1*05:03:18, DQB1*05:03:19, DQB1*05:03:20, DQB1*05:04, DQB1*05:05:01, DQB1*05:05:02, DQB1*05:06:01, DQB1*05:06:02, DQB1*05:07, DQB1*05:08, DQB1*05:09, DQB1*05:10, DQB1*05:100, DQB1*05:101, DQB1*05:102, DQB1*05:103, DQB1*05:104, DQB1*05:105, DQB1*05:106, DQB1*05:107, DQB1*05:108, DQB1*05:109, DQB1*05:110N, DQB1*05:111, DQB1*05:112, DQB1*05:113, DQB1*05:114, DQB1*05:115, DQB1*05:116, DQB1*05:117, DQB1*05:118, DQB1*05:119, DQB1*05:11:01, DQB1*05:11:02, DQB1*05:12, DQB1*05:120, DQB1*05:121, DQB1*05:122, DQB1*05:123, DQB1*05:124, DQB1*05:125, DQB1*05:126, DQB1*05:127, DQB1*05:128N, DQB1*05:129, DQB1*05:13, DQB1*05:130, DQB1*05:131, DQB1*05:132Q, DQB1*05:133, DQB1*05:134, DQB1*05:135, DQB1*05:136, DQB1*05:137, DQB1*05:138, DQB1*05:139, DQB1*05:14, DQB1*05:140, DQB1*05:141, DQB1*05:142, DQB1*05:143, DQB1*05:144, DQB1*05:145, DQB1*05:146, DQB1*05:147, DQB1*05:148, DQB1*05:149, DQB1*05:15, DQB1*05:150, DQB1*05:151, DQB1*05:152, DQB1*05:153, DQB1*05:154, DQB1*05:155, DQB1*05:156, DQB1*05:157, DQB1*05:158, DQB1*05:159, DQB1*05:16, DQB1*05:160, DQB1*05:161, DQB1*05:162, DQB1*05:163, DQB1*05:164, DQB1*05:165, DQB1*05:166, DQB1*05:167, DQB1*05:168, DQB1*05:169, DQB1*05:17, DQB1*05:170, DQB1*05:171, DQB1*05:172, DQB1*05:173, DQB1*05:174, DQB1*05:175, DQB1*05:176, DQB1*05:177, DQB1*05:178, DQB1*05:179, DQB1*05:18, DQB1*05:180, DQB1*05:181, DQB1*05:182, DQB1*05:183, DQB1*05:184, DQB1*05:185N, DQB1*05:186, DQB1*05:187, DQB1*05:188, DQB1*05:189, DQB1*05:19, DQB1*05:190, DQB1*05:191, DQB1*05:192, DQB1*05:193, DQB1*05:194, DQB1*05:195, DQB1*05:196, DQB1*05:197, DQB1*05:198, DQB1*05:199, DQB1*05:20, DQB1*05:200, DQB1*05:201, DQB1*05:202, DQB1*05:203, DQB1*05:204, DQB1*05:205, DQB1*05:206N, DQB1*05:207, DQB1*05:208N5 bp, DQB1*05:209, DQB1*05:21, DQB1*05:210, DQB1*05:211, DQB1*05:212, DQB1*05:213, DQB1*05:214, DQB1*05:215N, DQB1*05:216, DQB1*05:217, DQB1*05:22, DQB1*05:23, DQB1*05:24, DQB1*05:25, DQB1*05:26, DQB1*05:27, DQB1*05:28, DQB1*05:29, DQB1*05:30, DQB1*05:31, DQB1*05:32, DQB1*05:33, DQB1*05:34, DQB1*05:35, DQB1*05:36, DQB1*05:37, DQB1*05:38, DQB1*05:39, DQB1*05:40, DQB1*05:41N, DQB1*05:42, DQB1*05:43:01, DQB1*05:43:02, DQB1*05:44, DQB1*05:45, DQB1*05:46, DQB1*05:47, DQB1*05:48, DQB1*05:49, DQB1*05:50, DQB1*05:51, DQB1*05:52, DQB1*05:53, DQB1*05:54, DQB1*05:55, DQB1*05:56, DQB1*05:57, DQB1*05:58, DQB1*05:59, DQB1*05:60, DQB1*05:61, DQB1*05:62, DQB1*05:63, DQB1*05:64, DQB1*05:65, DQB1*05:66:01, DQB1*05:66:02, DQB1*05:67, DQB1*05:68, DQB1*05:69, DQB1*05:70, DQB1*05:71, DQB1*05:72, DQB1*05:73, DQB1*05:74, DQB1*05:75, DQB1*05:76, DQB1*05:77, DQB1*05:78, DQB1*05:79, DQB1*05:80, DQB1*05:81, DQB1*05:82, DQB1*05:83, DQB1*05:84, DQB1*05:85, DQB1*05:86, DQB1*05:87Q, DQB1*05:88, DQB1*05:89:01, DQB1*05:89:02, DQB1*05:90N, DQB1*05:91, DQB1*05:92, DQB1*05:93, DQB1*05:94, DQB1*05:95, DQB1*05:96, DQB1*05:97, DQB1*05:98, DQB1*05:99, DQB1*06:01:01:01, DQB1*06:01:01:02, DQB1*06:01:02, DQB1*06:01:03, DQB1*06:01:04, DQB1*06:01:05, DQB1*06:01:06, DQB1*06:01:07, DQB1*06:01:08, DQB1*06:01:09, DQB1*06:01:10, DQB1*06:01:11, DQB1*06:01:12, DQB1*06:01:13, DQB1*06:01:14, DQB1*06:01:15, DQB1*06:01:16, DQB1*06:01:17, DQB1*06:01:18, DQB1*06:01:19, DQB1*06:01:20, DQB1*06:01:21, DQB1*06:02:01:01, DQB1*06:02:01:02, DQB1*06:02:01:03, DQB1*06:02:01:04, DQB1*06:02:02, DQB1*06:02:03, DQB1*06:02:04, DQB1*06:02:05, DQB1*06:02:06, DQB1*06:02:07, DQB1*06:02:08, DQB1*06:02:09, DQB1*06:02:10, DQB1*06:02:11, DQB1*06:02:12, DQB1*06:02:13, DQB1*06:02:14, DQB1*06:02:15, DQB1*06:02:16, DQB1*06:02:17, DQB1*06:02:18, DQB1*06:02:19, DQB1*06:02:20, DQB1*06:02:21, DQB1*06:02:22, DQB1*06:02:23, DQB1*06:02:24, DQB1*06:02:25, DQB1*06:02:26, DQB1*06:02:27, DQB1*06:02:28, DQB1*06:02:29, DQB1*06:02:30, DQB1*06:02:31, DQB1*06:02:32, DQB1*06:02:33, DQB1*06:02:34, DQB1*06:02:35, DQB1*06:02:36, DQB1*06:02:37, DQB1*06:02:38, DQB1*06:03:01:01, DQB1*06:03:01:02, DQB1*06:03:01:03, DQB1*06:03:02, DQB1*06:03:03, DQB1*06:03:04, DQB1*06:03:05, DQB1*06:03:06, DQB1*06:03:07, DQB1*06:03:08, DQB1*06:03:09, DQB1*06:03:10, DQB1*06:03:11, DQB1*06:03:12, DQB1*06:03:13, DQB1*06:03:14, DQB1*06:03:15, DQB1*06:03:16, DQB1*06:03:17, DQB1*06:03:18, DQB1*06:03:19, DQB1*06:03:20, DQB1*06:03:21, DQB1*06:03:22, DQB1*06:03:23, DQB1*06:03:24, DQB1*06:03:25, DQB1*06:03:26, DQB1*06:03:27, DQB1*06:03:28, DQB1*06:03:29, DQB1*06:03:30, DQB1*06:03:31, DQB1*06:03:32, DQB1*06:03:33, DQB1*06:03:34, DQB1*06:03:35, DQB1*06:04:01, DQB1*06:04:02, DQB1*06:04:03, DQB1*06:04:04, DQB1*06:04:05, DQB1*06:04:06, DQB1*06:04:07, DQB1*06:04:08, DQB1*06:04:09, DQB1*06:04:10, DQB1*06:04:11, DQB1*06:04:12, DQB1*06:05:01, DQB1*06:05:02, DQB1*06:06, DQB1*06:07:01, DQB1*06:07:02, DQB1*06:08:01, DQB1*06:08:02, DQB1*06:08:03, DQB1*06:09:01:01, DQB1*06:09:01:02, DQB1*06:09:02, DQB1*06:09:03, DQB1*06:09:04, DQB1*06:09:05, DQB1*06:09:06, DQB1*06:09:07, DQB1*06:09:08, DQB1*06:09:09, DQB1*06:09:10, DQB1*06:10, DQB1*06:100, DQB1*06:101, DQB1*06:102N, DQB1*06:103, DQB1*06:104, DQB1*06:105, DQB1*06:106, DQB1*06:107, DQB1*06:108, DQB1*06:109, DQB1*06:110, DQB1*06:111, DQB1*06:112N, DQB1*06:113, DQB1*06:114, DQB1*06:115, DQB1*06:116, DQB1*06:117, DQB1*06:118:01, DQB1*06:118:02, DQB1*06:118:03, DQB1*06:119, DQB1*06:11:01, DQB1*06:11:02, DQB1*06:11:03, DQB1*06:11:04, DQB1*06:12, DQB1*06:120, DQB1*06:121, DQB1*06:122, DQB1*06:123, DQB1*06:124, DQB1*06:125, DQB1*06:126, DQB1*06:127, DQB1*06:128, DQB1*06:129, DQB1*06:130, DQB1*06:131, DQB1*06:132, DQB1*06:133, DQB1*06:134, DQB1*06:135, DQB1*06:136, DQB1*06:137, DQB1*06:138, DQB1*06:139, DQB1*06:13:01, DQB1*06:13:02, DQB1*06:13:03, DQB1*06:140, DQB1*06:141, DQB1*06:142, DQB1*06:143, DQB1*06:144N, DQB1*06:145, DQB1*06:146:01, DQB1*06:146:02, DQB1*06:147, DQB1*06:148, DQB1*06:149, DQB1*06:14:01, DQB1*06:14:02, DQB1*06:14:03, DQB1*06:150, DQB1*06:151, DQB1*06:152, DQB1*06:153:01, DQB1*06:153:02, DQB1*06:154, DQB1*06:155, DQB1*06:156, DQB1*06:157, DQB1*06:158N, DQB1*06:159, DQB1*06:15:01, DQB1*06:15:02, DQB1*06:16, DQB1*06:160, DQB1*06:161, DQB1*06:162, DQB1*06:163, DQB1*06:164, DQB1*06:165, DQB1*06:166, DQB1*06:167, DQB1*06:168, DQB1*06:169, DQB1*06:17, DQB1*06:170, DQB1*06:171, DQB1*06:172, DQB1*06:173, DQB1*06:174, DQB1*06:175, DQB1*06:176, DQB1*06:177, DQB1*06:178, DQB1*06:179N, DQB1*06:180, DQB1*06:181, DQB1*06:182, DQB1*06:183, DQB1*06:184, DQB1*06:185, DQB1*06:186, DQB1*06:187, DQB1*06:188, DQB1*06:189, DQB1*06:18:01, DQB1*06:18:02, DQB1*06:190:01, DQB1*06:190:02, DQB1*06:191, DQB1*06:192, DQB1*06:193N, DQB1*06:194, DQB1*06:195, DQB1*06:196, DQB1*06:197, DQB1*06:198, DQB1*06:199, DQB1*06:19:01, DQB1*06:19:02, DQB1*06:20, DQB1*06:200, DQB1*06:201, DQB1*06:202, DQB1*06:203, DQB1*06:204, DQB1*06:205, DQB1*06:206:01, DQB1*06:206:02, DQB1*06:207, DQB1*06:208, DQB1*06:209, DQB1*06:21, DQB1*06:210, DQB1*06:211, DQB1*06:212, DQB1*06:213, DQB1*06:214, DQB1*06:215, DQB1*06:216N, DQB1*06:217, DQB1*06:218, DQB1*06:219, DQB1*06:221, DQB1*06:222, DQB1*06:223, DQB1*06:224, DQB1*06:225, DQB1*06:226, DQB1*06:227, DQB1*06:228, DQB1*06:229, DQB1*06:22:01, DQB1*06:22:02, DQB1*06:22:03, DQB1*06:23, DQB1*06:230, DQB1*06:231, DQB1*06:232, DQB1*06:233, DQB1*06:234, DQB1*06:235, DQB1*06:236, DQB1*06:237, DQB1*06:238, DQB1*06:239, DQB1*06:24, DQB1*06:240, DQB1*06:241, DQB1*06:242, DQB1*06:243, DQB1*06:244, DQB1*06:245, DQB1*06:246, DQB1*06:247, DQB1*06:248, DQB1*06:249, DQB1*06:25, DQB1*06:250, DQB1*06:251, DQB1*06:252N, DQB1*06:253, DQB1*06:254, DQB1*06:255, DQB1*06:256, DQB1*06:257, DQB1*06:258, DQB1*06:259, DQB1*06:260, DQB1*06:261, DQB1*06:262, DQB1*06:263, DQB1*06:264, DQB1*06:265, DQB1*06:266, DQB1*06:267, DQB1*06:268, DQB1*06:269, DQB1*06:26N, DQB1*06:270:01, DQB1*06:270:02, DQB1*06:271, DQB1*06:272, DQB1*06:273, DQB1*06:274, DQB1*06:275, DQB1*06:276, DQB1*06:277, DQB1*06:278, DQB1*06:279, DQB1*06:27:01, DQB1*06:27:02, DQB1*06:28, DQB1*06:280, DQB1*06:281, DQB1*06:282, DQB1*06:283, DQB1*06:284, DQB1*06:285, DQB1*06:286, DQB1*06:287, DQB1*06:288, DQB1*06:289, DQB1*06:29, DQB1*06:290, DQB1*06:291, DQB1*06:292, DQB1*06:293, DQB1*06:294, DQB1*06:295, DQB1*06:296, DQB1*06:297, DQB1*06:298, DQB1*06:299, DQB1*06:30, DQB1*06:300, DQB1*06:301, DQB1*06:302, DQB1*06:303N, DQB1*06:304N, DQB1*06:305, DQB1*06:306N, DQB1*06:307, DQB1*06:308N, DQB1*06:309, DQB1*06:31, DQB1*06:310, DQB1*06:311, DQB1*06:312, DQB1*06:313, DQB1*06:314, DQB1*06:315, DQB1*06:316, DQB1*06:317N, DQB1*06:318, DQB1*06:319, DQB1*06:320, DQB1*06:321, DQB1*06:322, DQB1*06:323, DQB1*06:324, DQB1*06:325, DQB1*06:326, DQB1*06:32:01, DQB1*06:32:02, DQB1*06:33, DQB1*06:34, DQB1*06:35, DQB1*06:36, DQB1*06:37, DQB1*06:38, DQB1*06:39, DQB1*06:40, DQB1*06:41, DQB1*06:42, DQB1*06:43, DQB1*06:44, DQB1*06:45, DQB1*06:46, DQB1*06:47, DQB1*06:48:01, DQB1*06:48:02, DQB1*06:49, DQB1*06:50, DQB1*06:51:01, DQB1*06:51:02, DQB1*06:52, DQB1*06:53:01, DQB1*06:53:02, DQB1*06:54N, DQB1*06:55, DQB1*06:56, DQB1*06:57, DQB1*06:58, DQB1*06:59, DQB1*06:60, DQB1*06:61, DQB1*06:62, DQB1*06:63, DQB1*06:64, DQB1*06:65, DQB1*06:66, DQB1*06:67, DQB1*06:68, DQB1*06:69:01, DQB1*06:69:02, DQB1*06:70, DQB1*06:71, DQB1*06:72, DQB1*06:73, DQB1*06:74, DQB1*06:75NX, DQB1*06:76, DQB1*06:77N, DQB1*06:78, DQB1*06:79:01, DQB1*06:79:02, DQB1*06:80, DQB1*06:81, DQB1*06:82, DQB1*06:83, DQB1*06:84, DQB1*06:85, DQB1*06:86, DQB1*06:87, DQB1*06:88, DQB1*06:89, DQB1*06:90, DQB1*06:91, DQB1*06:92:01, DQB1*06:92:02, DQB1*06:93, DQB1*06:94, DQB1*06:95, DQB1*06:96:01, DQB1*06:96:02, DQB1*06:97, DQB1*06:98, DQB1*06:99:01, DQB1*06:99:02, and any combination thereof.

In certain aspects, the MHC class II molecule comprises a DQ beta chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 13, wherein the DQ beta chain comprises a tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 11, and wherein the DQ beta chain comprises a methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11. In certain aspects, the MHC class II molecule comprises a DQ beta chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 13, wherein the DQ beta chain comprises (i) a tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 11, (ii) a methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 11, (iii) a glutamine at a position corresponding to amino acid residue 110 of SEQ ID NO: 11; (iv) a valine at a position corresponding to amino acid residue 116 of SEQ ID NO: 11; (v) a histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 11; and (vi) a glutamine at a position corresponding to amino acid residue 146 of SEQ ID NO: 11. In certain aspects, the MHC class II molecule comprises a DQ beta chain comprising an amino acid sequence set forth in SEQ ID NO: 13.

II.A.2.b. HLA-DQ Alpha Chain

In some aspects of the present disclosure, the MHC class II molecule further comprises an alpha chain. In some aspects, the alpha chain is a wild-type alpha chain. In some aspects, the alpha chain is a DQ alpha chain. Any DQ alpha chain can be used in the compositions and methods of the present disclosure. In some aspects, the DQ alpha chain comprises an HLA-DQA1*01, HLA-DQA1*02, HLA-DQA1*03, HLA-DQA1*04, HLA-DQA1*05, or HLA-DQA1*06 allele. In certain aspects, the DQ alpha chain comprises an HLA-DQA1*01 allele. In certain aspects, the DQ alpha chain comprises an HLA-DQA1*02 allele. In certain aspects, the DQ alpha chain comprises an HLA-DQA1*03 allele. In certain aspects, the DQ alpha chain comprises an HLA-DQA1*04 allele. In certain aspects, the DQ alpha chain comprises an HLA-DQA1*05 allele. In certain aspects, the DQ alpha chain comprises an HLA-DQA1*06 allele.

In certain aspects, the DQ alpha chain is selected from DQA1*01:01:01:01, DQA1*01:01:01:02, DQA1*01:01:01:03, DQA1*01:01:01:05, DQA1*01:01:01:06, DQA1*01:01:02, DQA1*01:01:03, DQA1*01:01:04, DQA1*01:01:05, DQA1*01:02:01:01, DQA1*01:02:01:02, DQA1*01:02:01:03, DQA1*01:02:01:04, DQA1*01:02:01:05, DQA1*01:02:01:06, DQA1*01:02:01:07, DQA1*01:02:01:08, DQA1*01:02:01:09, DQA1*01:02:01:10, DQA1*01:02:01:11, DQA1*01:02:01:12, DQA1*01:02:02:01, DQA1*01:02:02:02, DQA1*01:02:02:03, DQA1*01:02:02:04, DQA1*01:02:03, DQA1*01:02:04, DQA1*01:03:01:01, DQA1*01:03:01:02, DQA1*01:03:01:03, DQA1*01:03:01:04, DQA1*01:03:01:05, DQA1*01:03:01:06, DQA1*01:03:01:07, DQA1*01:03:01:08, DQA1*01:03:01:09, DQA1*01:04:01:01, DQA1*01:04:01:02, DQA1*01:04:01:03, DQA1*01:04:01:04, DQA1*01:04:02, DQA1*01:05:01, DQA1*01:05:02, DQA1*01:06, DQA1*01:07Q, DQA1*01:08, DQA1*01:09, DQA1*01:10, DQA1*01:11, DQA1*01:12, DQA1*01:13, DQA1*01:14, DQA1*01:15N, DQA1*01:16N, DQA1*01:17, DQA1*01:18, DQA1*01:19, DQA1*01:20, DQA1*01:21, DQA1*01:22, DQA1*01:23, DQA1*01:24, DQA1*01:25, DQA1*01:26, DQA1*02:01:01:01, DQA1*02:01:01:02, DQA1*02:01:02, DQA1*02:02N, DQA1*02:03, DQA1*03:01:01, DQA1*03:01:03, DQA1*03:02:01:01, DQA1*03:02:01:02, DQA1*03:03:01:01, DQA1*03:03:01:02, DQA1*03:03:01:03, DQA1*03:03:01:04, DQA1*03:03:01:05, DQA1*03:03:01:06, DQA1*03:03:01:07, DQA1*03:03:02, DQA1*03:04, DQA1*03:05, DQA1*03:06, DQA1*03:07, DQA1*04:01:01:01, DQA1*04:01:01:02, DQA1*04:01:01:03, DQA1*04:01:01:04, DQA1*04:01:01:05, DQA1*04:01:01:06, DQA1*04:01:01:07, DQA1*04:01:01:08, DQA1*04:01:02:01, DQA1*04:01:02:02, DQA1*04:01:03, DQA1*04:02, DQA1*04:03N, DQA1*04:04, DQA1*04:05, DQA1*05:01:01:01, DQA1*05:01:01:02, DQA1*05:01:01:03, DQA1*05:01:01:04, DQA1*05:01:02, DQA1*05:01:04, DQA1*05:01:05, DQA1*05:01:06, DQA1*05:02, DQA1*05:03:01:01, DQA1*05:03:01:02, DQA1*05:04, DQA1*05:05:01:01, DQA1*05:05:01:02, DQA1*05:05:01:03, DQA1*05:05:01:04, DQA1*05:05:01:05, DQA1*05:05:01:06, DQA1*05:05:01:07, DQA1*05:05:01:08, DQA1*05:05:01:09, DQA1*05:05:01:10, DQA1*05:05:01:11, DQA1*05:05:01:12, DQA1*05:05:01:13, DQA1*05:05:01:14, DQA1*05:05:01:15, DQA1*05:05:01:16, DQA1*05:05:01:17, DQA1*05:05:01:18, DQA1*05:05:01:19, DQA1*05:05:01:20, DQA1*05:06:01:01, DQA1*05:06:01:02, DQA1*05:07, DQA1*05:08, DQA1*05:09, DQA1*05:10, DQA1*05:11, DQA1*05:12, DQA1*05:13, DQA1*05:14, DQA1*05:15N, DQA1*06:01:01:01, DQA1*06:01:01:02, DQA1*06:01:01:03, DQA1*06:01:01:04, DQA1*06:01:02, DQA1*06:02, and any combination thereof.

In certain aspects, the MHC class II molecule comprises a DQ alpha chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 16. In certain aspects, the MHC class II molecule comprises a DQ alpha chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 18. In certain aspects, the MHC class II molecule comprises a DQ alpha chain comprising an amino acid sequence set forth in SEQ ID NO: 16. In certain aspects, the MHC class II molecule comprises a DQ alpha chain comprising an amino acid sequence set forth in SEQ ID NO: 18.

II.A.3. HLA-DR Molecules

Many HLA-DR alleles are known in the art, and any of the known alleles can be used in the present disclosure. Examples of HLA-DR alpha chain and beta chain alleles are shown in Table 5. An updated list of HLA alleles is available at hla.alleles.org/(last visited on Jul. 10, 2019).

TABLE 5

DR Beta chain and alpha chain amino acid and nucleotide sequences.

Beta Chain

DRB1*01: 01 Extracellular Domain (SEQ ID NO: 19)

GDTRPRFLWQLKFECHFFNGTERVRLLERCIYNQEESVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQRRAA

VDTYCRHNYGVGESFTVQRRVEPKVTVYPSKTQPLQHHNLLVCSVSGFYPGSIEVRWFRNGQEEKAGVVSTGLI

QNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSK

DRB1*01: 01 Extracellular Domain (SEQ ID NO: 20)

GGGGACACCCGACCACGTTTCTTGTGGCAGCTTAAGTTTGAATGTCATTTCTTCAATGGGACGGAGCGGGTGCG

GTTGCTGGAAAGATGCATCTATAACCAAGAGGAGTCCGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGG

TGACGGAGCTGGGGCGGCCTGATGCCGAGTACTGGAACAGCCAGAAGGACCTCCTGGAGCAGAGGCGGGCCGCG

GTGGACACCTACTGCAGACACAACTACGGGGTTGGTGAGAGCTTCACAGTGCAGCGGCGAGTTGAGCCTAAGGT

GACTGTGTATCCTTCAAAGACCCAGCCCCTGCAGCACCACAACCTCCTGGTCTGCTCTGTGAGTGGTTTCTATC

CAGGCAGCATTGAAGTCAGGTGGTTCCGGAACGGCCAGGAAGAGAAGGCTGGGGTGGTGTCCACAGGCCTGATC

CAGAATGGAGATTGGACCTTCCAGACCCTGGTGATGCTGGAAACAGTTCCTCGGAGTGGAGAGGTTTACACCTG

CCAAGTGGAGCACCCAAGTGTGACGAGCCCTCTCACAGTGGAATGGAGAGCACGGTCTGAATCTGCACAGAGCA

AG

DRB1*01: 01 L114W/V143M Extracellular Domain (SEQ ID NO: 268)

GDTRPRFLWQLKFECHFFNGTERVRLLERCIYNQEESVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQRRAA

VDTYCRHNYGVGESFTVQRRVEPKVTVYPSKTQPLQHHNWLVCSVSGFYPGSIEVRWFRNGQEEKAGVMSTGLI

QNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSK

DRB1*01: 01 L114W/V143M + 6reps Extracellular Domain (SEQ ID NO: 269)

GDTRPRFLWQLKFECHFFNGTERVRLLERCIYNQEESVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQRRAA

VDTYCRHNYGVGESFTVQRRVEPKVTVYPSKTQPLQHHNWLVCHVSGFYPGSIEVRWFRNGQEETAGVMSTNLI

QNGDWTFQILVMLEMTPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSK

DRB1*01: 01 L114W/V143M/S118H/T157I Extracellular Domain (SEQ ID NO: 21)

GDTRPRFLWQLKFECHFFNGTERVRLLERCIYNQEESVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQRRAA

VDTYCRHNYGVGESFTVQRRVEPKVTVYPSKTQPLQHHNWLVCHVSGFYPGSIEVRWFRNGQEEKAGVMSTGLI

QNGDWTFQILVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSK

Signal Peptide; DRB1*01: 01 L114W/V143M/S118H/T157I Extracellular Domain;

Gly/Ser Linker; Zip Sequences and His tag sequences) (SEQ ID NO: 22)

MMRPIVLVLLFATSALA
GDTRPRFLWQLKFECHFFNGTERVRLLERCIYNQEESVRFDSDVGEYRAVTELGRPD

AEYWNSQKDLLEQRRAAVDTYCRHNYGVGESFTVQRRVEPKVTVYPSKTQPLQHHNWLVCHVSGFYPGSIEVRW

FRNGQEEKAGVMSTGLIQNGDWTFQILVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSK
GGGGSLE

IEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK

Full-length wild-type DRB1*01: 01 (SEQ ID NO: 23)

MVCLKLPGGSCMTALTVTLMVLSSPLALAGDTRPRFLWQLKFECHFFNGTERVRLLERCIYNQEESVRFDSDVG

EYRAVTELGRPDAEYWNSQKDLLEQRRAAVDTYCRHNYGVGESFTVQRRVEPKVTVYPSKTQPLQHHNLLVCSV

SGFYPGSIEVRWFRNGQEEKAGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSE

SAQSKMLSGVGGFVLGLLFLGAGLFIYFRNQKGHSGLQPTGFLS

Signal Peptide; DRB1*01: 01 Extracellular Domain; and Gly/Ser Linker, Zip

Sequences, and biotinylation sequences) (SEQ ID NO: 270)

MMRPIVLVLLFATSALAGDTRPRFLWQLKFECHFFNGTERVRLLERCIYNQEESVRFDSDVGEYRAVTELGRPD

AEYWNSQKDLLEQRRAAVDTYCRHNYGVGESFTVQRRVEPKVTVYPSKTQPLQHHNLLVCSVSGFYPGSIEVRW

FRNGQEEKAGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSKGGGGSLE

IEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGKGSGLNDIFEAQKIEWHE

Signal Peptide; DRB1*01: 01 Extracellular Domain; and Gly/Ser Linker, Zip

Sequences, and biotinylation sequences) (SEQ ID NO: 271)

ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTCGCCACATCTGCCCTGGCCGGGGACACCCGACCACGTTTCTT

GTGGCAGCTTAAGTTTGAATGTCATTTCTTCAATGGGACGGAGCGGGTGCGGTTGCTGGAAAGATGCATCTATA

ACCAAGAGGAGTCCGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGTGACGGAGCTGGGGCGGCCTGAT

GCCGAGTACTGGAACAGCCAGAAGGACCTCCTGGAGCAGAGGCGGGCCGCGGTGGACACCTACTGCAGACACAA

CTACGGGGTTGGTGAGAGCTTCACAGTGCAGCGGCGAGTTGAGCCTAAGGTGACTGTGTATCCTTCAAAGACCC

AGCCCCTGCAGCACCACAACCTCCTGGTCTGCTCTGTGAGTGGTTTCTATCCAGGCAGCATTGAAGTCAGGTGG

TTCCGGAACGGCCAGGAAGAGAAGGCTGGGGTGGTGTCCACAGGCCTGATCCAGAATGGAGATTGGACCTTCCA

GACCCTGGTGATGCTGGAAACAGTTCCTCGGAGTGGAGAGGTTTACACCTGCCAAGTGGAGCACCCAAGTGTGA

CGAGCCCTCTCACAGTGGAATGGAGAGCACGGTCTGAATCTGCACAGAGCAAGGGCGGCGGAGGCAGCCTGGAA

ATCGAGGCCGCCTTCCTGGAAAGAGAGAACACCGCCCTGGAAACCCGGGTGGCCGAGCTGAGACAGAGAGTGCA

GAGACTGCGGAACCGGGTGTCCCAGTACCGGACCAGATATGGCCCTCTGGGAGGCGGCAAAGGGTCCGGCTTGA

ACGACATTTTTGAGGCCCAGAAGATAGAGTGGCACGAGTGA

Signal Peptide; DRB1*01: 01 L114W/V143M Extracellular Domain; Gly/Ser Linker;

Zip Sequences and His tag sequences) (SEQ ID NO: 274)

MMRPIVLVLLFATSALAGDTRPRFLWQLKFECHFFNGTERVRLLERCIYNQEESVRFDSDVGEYRAVTELGRPD

AEYWNSQKDLLEQRRAAVDTYCRHNYGVGESFTVQRRVEPKVTVYPSKTQPLQHHNWLVCSVSGFYPGSIEVRW

FRNGQEEKAGVMSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSK
GGGGSLE

IEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK

Signal Peptide; DRB1*01: 01 L114W/V143M/S118H/T157I Extracellular Domain;

Gly/Ser Linker, Zip Sequences, and biotinylation sequences) (SEQ ID NO: 272)

MMRPIVLVLLFATSALAGDTRPRFLWQLKFECHFFNGTERVRLLERCIYNQEESVRFDSDVGEYRAVTELGRPD

AEYWNSQKDLLEQRRAAVDTYCRHNYGVGESFTVQRRVEPKVTVYPSKTQPLQHHNWLVCHVSGFYPGSIEVRW

FRNGQEEKAGVMSTGLIQNGDWTFQILVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSKGGGGSLE

IEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGKGSGLNDIFEAQKIEWHE

Signal Peptide; DRB1*01: 01 L114W/V143M/S118H/T157I Extracellular Domain;

Gly/Ser Linker, Zip Sequences, and biotinylation sequences) (SEQ ID NO: 273)

ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTCGCCACATCTGCCCTGGCCGGGGACACCCGACCACGTTTCTT

GTGGCAGCTTAAGTTTGAATGTCATTTCTTCAATGGGACGGAGCGGGTGCGGTTGCTGGAAAGATGCATCTATA

ACCAAGAGGAGTCCGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGTGACGGAGCTGGGGCGGCCTGAT

GCCGAGTACTGGAACAGCCAGAAGGACCTCCTGGAGCAGAGGCGGGCCGCGGTGGACACCTACTGCAGACACAA

CTACGGGGTTGGTGAGAGCTTCACAGTGCAGCGGCGAGTTGAGCCTAAGGTGACTGTGTATCCTTCAAAGACCC

AGCCCCTGCAGCACCACAACTGGCTGGTCTGCCATGTGAGTGGTTTCTATCCAGGCAGCATTGAAGTCAGGTGG

TTCCGGAACGGCCAGGAAGAGAAGGCTGGGGTGATGTCCACAGGCCTGATCCAGAATGGAGATTGGACCTTCCA

GATCCTGGTGATGCTGGAAACAGTTCCTCGGAGTGGAGAGGTTTACACCTGCCAAGTGGAGCACCCAAGTGTGA

CGAGCCCTCTCACAGTGGAATGGAGAGCACGGTCTGAATCTGCACAGAGCAAGGGCGGCGGAGGCAGCCTGGAA

ATCGAGGCCGCCTTCCTGGAAAGAGAGAACACCGCCCTGGAAACCCGGGTGGCCGAGCTGAGACAGAGAGTGCA

GAGACTGCGGAACCGGGTGTCCCAGTACCGGACCAGATATGGCCCTCTGGGAGGCGGCAAAGGGTCCGGCTTGA

ACGACATTTTTGAGGCCCAGAAGATAGAGTGGCACGAGTGA

Signal Peptide; DRB1*01: 01 L114W/V143M + 6rps Extracellular Domain; Gly/Ser

Linker; Zip Sequences and His tag sequences) (SEQ ID NO: 275)

MMRPIVLVLLFATSALAGDTRPRFLWQLKFECHFFNGTERVRLLERCIYNQEESVRFDSDVGEYRAVTELGRPD

AEYWNSQKDLLEQRRAAVDTYCRHNYGVGESFTVQRRVEPKVTVYPSKTQPLQHHNWLVCHVSGFYPGSIEVRW

FRNGQEETAGVMSTNLIQNGDWTFQILVMLEMTPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSK
GGGGSLE

IEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK

Alpha Chain

DRA1*01: 01 Extracellular Domain (SEQ ID NO: 24)

IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAVDKANLEIMT

KRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTTGVSETVFLPREDHLFRKF

HYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPETTEN

DRA1*01: 01 Extracellular Domain (SEQ ID NO: 25)

ATCAAAGAAGAACATGTGATCATCCAGGCCGAGTTCTATCTGAATCCTGACCAATCAGGCGAGTTTATGTTTGA

CTTTGATGGTGATGAGATTTTCCATGTGGATATGGCAAAGAAGGAGACGGTCTGGCGGCTTGAAGAATTTGGAC

GATTTGCCAGCTTTGAGGCTCAAGGTGCATTGGCCAACATAGCTGTGGACAAAGCCAACCTGGAAATCATGACA

AAGCGCTCCAACTATACTCCGATCACCAATGTACCTCCAGAGGTAACTGTGCTCACAAACAGCCCTGTGGAACT

GAGAGAGCCCAACGTCCTCATCTGTTTCATAGACAAGTTCACCCCACCAGTGGTCAATGTCACGTGGCTTCGAA

ATGGAAAACCTGTCACCACAGGAGTGTCAGAGACAGTCTTCCTGCCCAGGGAAGACCACCTTTTCCGCAAGTTC

CACTATCTCCCCTTCCTGCCCTCAACTGAGGACGTTTACGACTGCAGGGTGGAGCACTGGGGCTTGGATGAGCC

TCTTCTCAAGCACTGGGAGTTTGATGCTCCAAGCCCTCTCCCAGAGACTACAGAGAAC

Signal Peptide; DRA1*01: 01 Extracellular Domain; Gly/Ser Linker, Zip

Sequences and His tag sequences)(SEQ ID NO: 26)

MMRPIVLVLLFATSALA
IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQ

GALANIAVDKANLEIMTKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTTG

VSETVFLPREDHLFRKEHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPETTEN
GGGGSLEIRAAFL

RQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGKGSHHHHHH

Signal Peptide; DRA1*01: 01 Extracellular Domain; Gly/Ser Linker, Zip

Sequences and His tag sequences (10x) (SEQ ID NO: 276)

MMRPIVLVLLFATSALA
IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQ

GALANIAVDKANLEIMTKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTTG

VSETVFLPREDHLFRKEHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPETTEN
GGGGSLEIRAAFL

RQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGKGSHHHHHHHHHH

Signal Peptide; DRA1*01: 01 Extracellular Domain; Gly/Ser Linker, Zip

Sequences and His tag sequences (10x) (SEQ ID NO: 277)

ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTCGCCACATCTGCCCTGGCC
ATCAAAGAAGAACATGTGATCAT

CCAGGCCGAGTTCTATCTGAATCCTGACCAATCAGGCGAGTTTATGTTTGACTTTGATGGTGATGAGATTTTCC

ATGTGGATATGGCAAAGAAGGAGACGGTCTGGCGGCTTGAAGAATTTGGACGATTTGCCAGCTTTGAGGCTCAA

GGTGCATTGGCCAACATAGCTGTGGACAAAGCCAACCTGGAAATCATGACAAAGCGCTCCAACTATACTCCGAT

CACCAATGTACCTCCAGAGGTAACTGTGCTCACGAACAGCCCTGTGGAACTGAGAGAGCCCAACGTCCTCATCT

GTTTCATCGACAAGTTCACCCCACCAGTGGTCAATGTCACGTGGCTTCGAAATGGAAAACCTGTCACCACAGGA

GTGTCAGAGACAGTCTTCCTGCCCAGGGAAGACCACCTTTTCCGCAAGTTCCACTATCTCCCCTTCCTGCCCTC

AACTGAGGACGTTTACGACTGCAGGGTGGAGCACTGGGGCTTGGATGAGCCTCTTCTCAAGCACTGGGAGTTTG

ATGCTCCAAGCCCTCTCCCAGAGACTACAGAGAAC
GGCGGCGGAGGCAGCCTGGAAATCAGAGCCGCCTTCCTG

CGGCAGAGAAACACCGCCCTGAGAACCGAAGTGGCCGAGCTGGAACAGGAAGTGCAGCGGCTGGAAAACGAGGT

GTCCCAGTACGAGACAAGATACGGCCCTCTGGGAGGCGGCAAGGGCTCTCACCACCACCATCACCATCATCATC

ACCATTGA

II.A.3.a. HLA-DR Beta Chain

In certain aspects, the HLA class II molecule comprises a DR beta chain, wherein the DR beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19. Any amino acid other than leucine can be present at the position corresponding to amino acid residue 114 of SEQ ID NO: 19. In some aspects, the amino acid other than leucine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 is an amino acid selected from an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 is an alanine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 is a valine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 is an isoleucine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 is a methionine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 is a phenylalanine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 is a tyrosine. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 is a tryptophan.

In some embodiments, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

In certain aspects, the HLA class II molecule comprises a DR beta chain, wherein the DR beta chain comprises an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19. Any amino acid other than valine can be present at the position corresponding to amino acid residue 143 of SEQ ID NO: 19. In some aspects, the amino acid other than valine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 19 is an amino acid selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 19 is an alanine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 19 is an isoleucine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 19 is a leucine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 19 is a methionine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 19 is a phenylalanine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 19 is a tyrosine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 19 is a tryptophan.

In some aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 19 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 143 of SEQ ID NO: 19 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

In certain aspects, the HLA class II molecule comprises a DR beta chain, wherein the DR beta chain comprises an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19. Any amino acid other than serine can be present at the position corresponding to amino acid residue 118 of SEQ ID NO: 19. In some aspects, the amino acid other than serine is an amino acid comprising an electrically charged side chain. In certain aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 19 is an amino acid selected from an arginine, a histidine, and a lysine. In certain aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 19 is an arginine. In certain aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 19 is a histidine. In certain aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 19 is a lysine.

In some aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 19 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises an electrically charged side chain. In certain aspects, the amino acid other than serine at the position corresponding to amino acid residue 118 of SEQ ID NO: 19 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises an electrically charged side chain.

In certain aspects, the HLA class II molecule comprises a DR beta chain, wherein the DR beta chain comprises an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19. Any amino acid other than threonine can be present at the position corresponding to amino acid residue 157 of SEQ ID NO: 19. In some aspects, the amino acid other than threonine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 19 is an amino acid selected an alanine, a valine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 19 is an alanine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 19 is a valine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 19 is an isoleucine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 19 is a leucine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 19 is a methionine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 19 is a phenylalanine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 19 is a tyrosine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 19 is a tryptophan.

In some aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 19 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 157 of SEQ ID NO: 19 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

In certain aspects, the HLA class II molecule comprises a DR beta chain, wherein the DR beta chain comprises an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19. Any amino acid other than lysine can be present at the position corresponding to amino acid residue 139 of SEQ ID NO: 19. In some aspects, the amino acid other than lysine is an amino acid comprising a polar uncharged side chain. In certain aspects, the amino acid other than lysine at the position corresponding to amino acid residue 139 of SEQ ID NO: 19 is an amino acid selected from a serine, a threonine, and a glutamine. In certain aspects, the amino acid other than lysine at the position corresponding to amino acid residue 139 of SEQ ID NO: 19 is a serine. In certain aspects, the amino acid other than lysine at the position corresponding to amino acid residue 139 of SEQ ID NO: 19 is a threonine. In certain aspects, the amino acid other than lysine at the position corresponding to amino acid residue 139 of SEQ ID NO: 19 is a glutamine.

In some aspects, the amino acid other than lysine at the position corresponding to amino acid residue 139 of SEQ ID NO: 19 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a polar uncharged side chain. In certain aspects, the amino acid other than lysine at the position corresponding to amino acid residue 139 of SEQ ID NO: 19 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a polar uncharged side chain.

In certain aspects, the HLA class II molecule comprises a DR beta chain, wherein the DR beta chain comprises an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19. Any amino acid other than glycine can be present at the position corresponding to amino acid residue 146 of SEQ ID NO: 19. In some aspects, the amino acid other than glycine is an amino acid comprising a polar uncharged side chain. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 19 is an amino acid selected from a serine, an asparagine, a threonine, and a glutamine. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 19 is a serine. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 19 is an asparagine. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 19 is a threonine. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 19 is a glutamine.

In some aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 19 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a polar uncharged side chain. In certain aspects, the amino acid other than glycine at the position corresponding to amino acid residue 146 of SEQ ID NO: 19 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a polar uncharged side chain.

In certain aspects, the HLA class II molecule comprises a DR beta chain, wherein the DR beta chain comprises an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19. Any amino acid other than threonine can be present at the position corresponding to amino acid residue 163 of SEQ ID NO: 19. In some aspects, the amino acid other than threonine is an amino acid comprising a hydrophobic side chain. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 19 is an amino acid selected from an alanine, a valine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 19 is an alanine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 19 is a valine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 19 is an isoleucine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 19 is a leucine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 19 is a methionine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 19 is a phenylalanine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 19 is a tyrosine. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 19 is a tryptophan.

In some aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 19 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a hydrophobic side chain. In certain aspects, the amino acid other than threonine at the position corresponding to amino acid residue 163 of SEQ ID NO: 19 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a hydrophobic side chain.

In certain aspects, the HLA class II molecule comprises a DR beta chain, wherein the DR beta chain comprises an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19. Any amino acid other than valine can be present at the position corresponding to amino acid residue 164 of SEQ ID NO: 19. In some aspects, the amino acid other than valine is an amino acid comprising a polar uncharged side chain. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 19 is an amino acid selected from a serine, an asparagine, a threonine, and a glutamine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 19 is a serine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 19 is an asparagine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 19 is a threonine. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 19 is a glutamine.

In some aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 19 consists of more than one amino acid, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some aspects at least one of the more than one amino acids comprises a polar uncharged side chain. In certain aspects, the amino acid other than valine at the position corresponding to amino acid residue 164 of SEQ ID NO: 19 consists of a series, e.g., at least 2, at least 3, at least 4, or at least 5, amino acids, wherein each of the series of amino acids comprises a polar uncharged side chain.

In certain aspects of the present disclosure, the MHC class II molecule comprises a DR beta chain comprising more than one substitution mutation relative to the wild-type DR beta chain. In certain aspects, the DR beta chain comprises at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, at least eight mutations, at least nine mutations, or at least ten mutations relative to the wild-type DR beta chain.

In certain aspects, the DR beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19 and an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19.

In certain aspects, the DR beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19; an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19; and at least two of: (i) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19, (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19.

In certain aspects, the DR beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19; an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19; and at least three of: (i) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19, (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19.

In certain aspects, the DR beta chain comprises an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19; an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19; and at least four of: (i) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, (ii) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (iii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (iv) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19, (v) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, and (vi) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19.

In certain aspects, the DR beta chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19, (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19, (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, and (d) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19; and at least one of: (i) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (ii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (iii) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, and (iv) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19.

In certain aspects, the DR beta chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19, (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19, (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, and (d) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19; and at least two of: (i) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (ii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (iii) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, and (iv) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19.

In certain aspects, the DR beta chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19, (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19, (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, and (d) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19; and at least three of: (i) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (ii) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (iii) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, and (iv) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19.

In certain aspects, the DR beta chain comprises (a) an amino acid other than leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19; (b) an amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19; (c) an amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, (d) an amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19, (e) an amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19, (f) an amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19, (g) an amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19, and (h) an amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19.

In certain aspects, the DR beta chain comprises (a) a tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 19, (b) a methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19, (c) a histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, and (d) an isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19.

In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19, (ii) the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19, or each of the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 and the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19 is an amino acid comprising a hydrophobic side chain.

In some aspects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 114 of SEQ ID NO: 19 is selected from an alanine, a valine, an isoleucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan; (ii) the amino acid other than valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19 is selected from an alanine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan; (iii) the amino acid other than serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19 is selected from an arginine, a histidine, and a lysine; (iv) the amino acid other than threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19 is selected from an alanine, a valine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan; (v) the amino acid other than lysine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19 is selected from a serine, a threonine, and a glutamine; (vi) the amino acid other than glycine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19 is selected from a serine, an asparagine, a threonine, and a glutamine; (vii) the amino acid other than threonine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19 is selected from an alanine, a valine, an isoleucine, a leucine, a methionine, a phenylalanine, a tyrosine, and a tryptophan; and/or (viii) the amino acid other than valine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19 is selected from a serine, an asparagine, a threonine, and a glutamine.

In certain aspects, a DR beta chain described herein has an increased affinity for a CD4 protein as compared to a reference HLA class II molecule. In some aspects, the reference HLA class II molecule is an HLA class II molecule having a wild-type DR beta chain. In some aspects, the reference HLA class II molecule is an HLA class II molecule having a DR beta chain comprising (i) a leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19 and/or (ii) a valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19. In some aspects, the reference HLA class II molecule is an HLA class II molecule having a DR beta chain comprising (i) a leucine at a position corresponding to amino acid residue 114 of SEQ ID NO: 19, (ii) a valine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19, (iii) a serine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19, and (iv) a threonine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19.

In certain aspects, the DR beta chain comprises an allele selected from an HLA-DRB1*01, an HLA-DRB1*03, an HLA-DRB1*04, an HLA-DRB1*06, an HLA-DRB1*07, an HLA-DRB1*08, an HLA-DRB1*09, an HLA-DRB1*10, an HLA-DRB1*11, an HLA-DRB1*12, an HLA-DRB1*13, an HLA-DRB1*14, an HLA-DRB1*15, or an HLA-DRB1*16 allele. In some aspects, the DR beta chain comprises an HLA-DRB1*01 allele. In particular aspects, the DR beta chain comprises an HLA-DRB1*01:01 allele.

In certain aspects, the DR beta chain comprises an allele selected from DRB1*01:01:01, DRB1*01:01:02, DRB1*01:01:03, DRB1*01:01:04, DRB1*01:01:05, DRB1*01:01:06, DRB1*01:01:07, DRB1*01:01:08, DRB1*01:01:09, DRB1*01:01:10, DRB1*01:01:11, DRB1*01:01:12, DRB1*01:01:13, DRB1*01:01:14, DRB1*01:01:15, DRB1*01:01:16, DRB1*01:01:17, DRB1*01:01:18, DRB1*01:01:19, DRB1*01:01:20, DRB1*01:01:21, DRB1*01:01:22, DRB1*01:01:23, DRB1*01:01:24, DRB1*01:01:25, DRB1*01:01:26, DRB1*01:01:27, DRB1*01:01:28, DRB1*01:01:29, DRB1*01:01:30, DRB1*01:01:31, DRB1*01:01:32, DRB1*01:01:33, DRB1*01:02:01:01, DRB1*01:02:01:02, DRB1*01:02:02, DRB1*01:02:03, DRB1*01:02:04, DRB1*01:02:05, DRB1*01:02:06, DRB1*01:02:07, DRB1*01:02:08, DRB1*01:02:09, DRB1*01:02:10, DRB1*01:02:11, DRB1*01:02:12, DRB1*01:02:13, DRB1*01:03:01, DRB1*01:03:02, DRB1*01:03:03, DRB1*01:03:04, DRB1*01:04, DRB1*01:05, DRB1*01:06, DRB1*01:07, DRB1*01:08, DRB1*01:09, DRB1*01:10, DRB1*01:100, DRB1*01:11:01, DRB1*01:11:02, DRB1*01:12, DRB1*01:13, DRB1*01:14, DRB1*01:15, DRB1*01:16, DRB1*01:17, DRB1*01:18:01, DRB1*01:18:02, DRB1*01:19, DRB1*01:20:01, DRB1*01:20:02, DRB1*01:21, DRB1*01:22, DRB1*01:23, DRB1*01:24:01, DRB1*01:24:02, DRB1*01:25, DRB1*01:26, DRB1*01:27, DRB1*01:28, DRB1*01:29:01, DRB1*01:29:02, DRB1*01:30, DRB1*01:31, DRB1*01:32, DRB1*01:33N, DRB1*01:34, DRB1*01:35, DRB1*01:36, DRB1*01:37, DRB1*01:38, DRB1*01:39N, DRB1*01:40N, DRB1*01:41, DRB1*01:42, DRB1*01:43, DRB1*01:44:01, DRB1*01:44:02, DRB1*01:45, DRB1*01:46, DRB1*01:47, DRB1*01:48, DRB1*01:49, DRB1*01:50, DRB1*01:51, DRB1*01:52N, DRB1*01:53, DRB1*01:54, DRB1*01:55, DRB1*01:56, DRB1*01:57, DRB1*01:58, DRB1*01:59, DRB1*01:60, DRB1*01:61, DRB1*01:62N, DRB1*01:63, DRB1*01:64, DRB1*01:65:01, DRB1*01:65:02, DRB1*01:66, DRB1*01:67, DRB1*01:68N, DRB1*01:69, DRB1*01:70, DRB1*01:71, DRB1*01:72, DRB1*01:73, DRB1*01:74, DRB1*01:75, DRB1*01:76, DRB1*01:77, DRB1*01:78, DRB1*01:79, DRB1*01:80, DRB1*01:81, DRB1*01:82, DRB1*01:83, DRB1*01:84, DRB1*01:85, DRB1*01:86, DRB1*01:87, DRB1*01:88, DRB1*01:89, DRB1*01:90, DRB1*01:91Q, DRB1*01:92, DRB1*01:93, DRB1*01:94, DRB1*01:95, DRB1*01:96, DRB1*01:97, DRB1*01:98, DRB1*01:99, DRB1*03:01:01:01, DRB1*03:01:01:02, DRB1*03:01:01:03, DRB1*03:01:02, DRB1*03:01:03, DRB1*03:01:04, DRB1*03:01:05, DRB1*03:01:06, DRB1*03:01:07, DRB1*03:01:08, DRB1*03:01:09, DRB1*03:01:10, DRB1*03:01:11, DRB1*03:01:12, DRB1*03:01:13, DRB1*03:01:14, DRB1*03:01:15, DRB1*03:01:16, DRB1*03:01:17, DRB1*03:01:18, DRB1*03:01:19, DRB1*03:01:20, DRB1*03:01:21, DRB1*03:01:22, DRB1*03:01:23, DRB1*03:01:24, DRB1*03:01:25, DRB1*03:01:26, DRB1*03:01:27, DRB1*03:01:28, DRB1*03:02:01, DRB1*03:02:02, DRB1*03:02:03, DRB1*03:03, DRB1*03:04:01, DRB1*03:04:02, DRB1*03:05:01, DRB1*03:05:02, DRB1*03:05:03, DRB1*03:06, DRB1*03:07:01, DRB1*03:07:02, DRB1*03:08, DRB1*03:09, DRB1*03:10, DRB1*03:100:01, DRB1*03:100:02, DRB1*03:101, DRB1*03:102, DRB1*03:103, DRB1*03:104, DRB1*03:105, DRB1*03:106, DRB1*03:107, DRB1*03:108, DRB1*03:109, DRB1*03:110, DRB1*03:111, DRB1*03:112, DRB1*03:113, DRB1*03:114, DRB1*03:115, DRB1*03:116, DRB1*03:117, DRB1*03:118, DRB1*03:119, DRB1*03:11:01, DRB1*03:12, DRB1*03:120, DRB1*03:121, DRB1*03:122, DRB1*03:123, DRB1*03:124, DRB1*03:125, DRB1*03:126, DRB1*03:127, DRB1*03:128, DRB1*03:129, DRB1*03:130, DRB1*03:131, DRB1*03:132, DRB1*03:133, DRB1*03:134, DRB1*03:135, DRB1*03:136, DRB1*03:137, DRB1*03:138, DRB1*03:139, DRB1*03:13:01, DRB1*03:13:02, DRB1*03:14, DRB1*03:140, DRB1*03:141, DRB1*03:142, DRB1*03:143, DRB1*03:144, DRB1*03:145, DRB1*03:146, DRB1*03:147, DRB1*03:148, DRB1*03:149, DRB1*03:150, DRB1*03:151, DRB1*03:152, DRB1*03:153, DRB1*03:154, DRB1*03:155, DRB1*03:156N, DRB1*03:157, DRB1*03:158, DRB1*03:15:01, DRB1*03:15:02, DRB1*03:16, DRB1*03:17, DRB1*03:18, DRB1*03:19, DRB1*03:20, DRB1*03:21, DRB1*03:22, DRB1*03:23, DRB1*03:24, DRB1*03:25:01, DRB1*03:25:02, DRB1*03:26, DRB1*03:27, DRB1*03:28, DRB1*03:29, DRB1*03:30, DRB1*03:31, DRB1*03:32, DRB1*03:33, DRB1*03:34, DRB1*03:35, DRB1*03:36, DRB1*03:37, DRB1*03:38, DRB1*03:39, DRB1*03:40, DRB1*03:41:01, DRB1*03:41:02, DRB1*03:42, DRB1*03:43, DRB1*03:44, DRB1*03:45, DRB1*03:46, DRB1*03:47, DRB1*03:48, DRB1*03:49, DRB1*03:50, DRB1*03:51, DRB1*03:52, DRB1*03:53, DRB1*03:54, DRB1*03:55, DRB1*03:56, DRB1*03:57, DRB1*03:58, DRB1*03:59, DRB1*03:60, DRB1*03:61, DRB1*03:62, DRB1*03:63, DRB1*03:64, DRB1*03:65, DRB1*03:66, DRB1*03:67N, DRB1*03:68N, DRB1*03:69, DRB1*03:70, DRB1*03:71:01, DRB1*03:71:02, DRB1*03:72, DRB1*03:73, DRB1*03:74, DRB1*03:75, DRB1*03:76, DRB1*03:77, DRB1*03:78, DRB1*03:79, DRB1*03:80, DRB1*03:81, DRB1*03:82, DRB1*03:83, DRB1*03:84, DRB1*03:85, DRB1*03:86, DRB1*03:87, DRB1*03:88, DRB1*03:89, DRB1*03:90, DRB1*03:91, DRB1*03:92, DRB1*03:93, DRB1*03:94, DRB1*03:95, DRB1*03:96, DRB1*03:97, DRB1*03:98, DRB1*03:99, DRB1*04:01:01:01, DRB1*04:01:01:02, DRB1*04:01:01:03, DRB1*04:01:02, DRB1*04:01:03, DRB1*04:01:04, DRB1*04:01:05, DRB1*04:01:06, DRB1*04:01:07, DRB1*04:01:08, DRB1*04:01:09, DRB1*04:01:10, DRB1*04:01:11, DRB1*04:01:12, DRB1*04:01:13, DRB1*04:01:14, DRB1*04:01:15, DRB1*04:01:16, DRB1*04:01:17, DRB1*04:01:18, DRB1*04:01:19, DRB1*04:01:20, DRB1*04:01:21, DRB1*04:02:01, DRB1*04:02:02, DRB1*04:02:03, DRB1*04:02:04, DRB1*04:02:05, DRB1*04:02:06, DRB1*04:03:01:01, DRB1*04:03:01:02, DRB1*04:03:02, DRB1*04:03:03, DRB1*04:03:04, DRB1*04:03:05, DRB1*04:03:06, DRB1*04:03:07, DRB1*04:03:08, DRB1*04:03:09, DRB1*04:03:10, DRB1*04:03:11, DRB1*04:03:12, DRB1*04:03:13, DRB1*04:03:14, DRB1*04:03:15, DRB1*04:04:01, DRB1*04:04:02, DRB1*04:04:03, DRB1*04:04:04, DRB1*04:04:05, DRB1*04:04:06, DRB1*04:04:07, DRB1*04:04:08, DRB1*04:04:09, DRB1*04:04:10, DRB1*04:04:11, DRB1*04:04:12, DRB1*04:04:13, DRB1*04:04:14, DRB1*04:04:15, DRB1*04:05:01:01, DRB1*04:05:01:02, DRB1*04:05:01:03, DRB1*04:05:02, DRB1*04:05:03, DRB1*04:05:04, DRB1*04:05:05, DRB1*04:05:06, DRB1*04:05:07, DRB1*04:05:08, DRB1*04:05:09, DRB1*04:05:10, DRB1*04:05:11, DRB1*04:05:13, DRB1*04:05:14, DRB1*04:05:15, DRB1*04:05:16, DRB1*04:05:17, DRB1*04:05:18, DRB1*04:05:19, DRB1*04:05:20, DRB1*04:06:01, DRB1*04:06:02, DRB1*04:06:03, DRB1*04:06:04, DRB1*04:06:05, DRB1*04:06:06, DRB1*04:06:07, DRB1*04:07:01:01, DRB1*04:07:01:02, DRB1*04:07:02, DRB1*04:07:03, DRB1*04:07:04, DRB1*04:07:05, DRB1*04:07:06, DRB1*04:08:01, DRB1*04:08:02, DRB1*04:08:03, DRB1*04:08:04, DRB1*04:09, DRB1*04:100, DRB1*04:101, DRB1*04:102, DRB1*04:103, DRB1*04:104, DRB1*04:105:01, DRB1*04:105:02, DRB1*04:106, DRB1*04:107, DRB1*04:108, DRB1*04:109, DRB1*04:10:01, DRB1*04:10:02, DRB1*04:10:03, DRB1*04:110, DRB1*04:111, DRB1*04:112, DRB1*04:113, DRB1*04:114, DRB1*04:115, DRB1*04:116, DRB1*04:117, DRB1*04:118, DRB1*04:119N, DRB1*04:11:01, DRB1*04:11:02, DRB1*04:11:03, DRB1*04:11:04, DRB1*04:11:05, DRB1*04:12, DRB1*04:120N, DRB1*04:121, DRB1*04:122, DRB1*04:123, DRB1*04:124, DRB1*04:125, DRB1*04:126, DRB1*04:127, DRB1*04:128, DRB1*04:129, DRB1*04:13, DRB1*04:130, DRB1*04:131:01, DRB1*04:131:02, DRB1*04:132, DRB1*04:133, DRB1*04:134, DRB1*04:135, DRB1*04:136, DRB1*04:137, DRB1*04:138, DRB1*04:139, DRB1*04:14, DRB1*04:140, DRB1*04:141, DRB1*04:142N, DRB1*04:143, DRB1*04:144, DRB1*04:145, DRB1*04:146, DRB1*04:147, DRB1*04:148, DRB1*04:149, DRB1*04:15, DRB1*04:150, DRB1*04:151, DRB1*04:152, DRB1*04:153, DRB1*04:154, DRB1*04:155, DRB1*04:156, DRB1*04:157N, DRB1*04:158N, DRB1*04:159, DRB1*04:16, DRB1*04:160, DRB1*04:161, DRB1*04:162, DRB1*04:163, DRB1*04:164, DRB1*04:165, DRB1*04:166, DRB1*04:167, DRB1*04:168, DRB1*04:169, DRB1*04:170, DRB1*04:171, DRB1*04:172, DRB1*04:173, DRB1*04:174, DRB1*04:175, DRB1*04:176, DRB1*04:177, DRB1*04:178N, DRB1*04:179, DRB1*04:17:01, DRB1*04:17:02, DRB1*04:18, DRB1*04:180, DRB1*04:181, DRB1*04:182, DRB1*04:183, DRB1*04:184, DRB1*04:185, DRB1*04:186N, DRB1*04:187, DRB1*04:188, DRB1*04:189, DRB1*04:19, DRB1*04:190, DRB1*04:191, DRB1*04:192, DRB1*04:193, DRB1*04:194, DRB1*04:195, DRB1*04:196, DRB1*04:197, DRB1*04:198, DRB1*04:199, DRB1*04:20, DRB1*04:200, DRB1*04:201, DRB1*04:202, DRB1*04:203, DRB1*04:204, DRB1*04:205, DRB1*04:206, DRB1*04:207, DRB1*04:208, DRB1*04:209, DRB1*04:21, DRB1*04:210, DRB1*04:211, DRB1*04:212N, DRB1*04:213, DRB1*04:214N, DRB1*04:215, DRB1*04:216, DRB1*04:217, DRB1*04:218, DRB1*04:219, DRB1*04:22, DRB1*04:220, DRB1*04:221, DRB1*04:222, DRB1*04:223, DRB1*04:224, DRB1*04:225, DRB1*04:226:01, DRB1*04:226:02, DRB1*04:227, DRB1*04:228, DRB1*04:229, DRB1*04:23, DRB1*04:230, DRB1*04:231, DRB1*04:232, DRB1*04:233, DRB1*04:234, DRB1*04:235, DRB1*04:236, DRB1*04:237, DRB1*04:238, DRB1*04:239, DRB1*04:24, DRB1*04:240, DRB1*04:241, DRB1*04:242, DRB1*04:243, DRB1*04:244, DRB1*04:245, DRB1*04:246, DRB1*04:247N, DRB1*04:248, DRB1*04:249, DRB1*04:25, DRB1*04:250, DRB1*04:251, DRB1*04:252, DRB1*04:253, DRB1*04:254, DRB1*04:255, DRB1*04:256, DRB1*04:257, DRB1*04:258, DRB1*04:259, DRB1*04:26, DRB1*04:260, DRB1*04:261, DRB1*04:262, DRB1*04:263, DRB1*04:264N, DRB1*04:265, DRB1*04:266N, DRB1*04:267N, DRB1*04:268, DRB1*04:269, DRB1*04:27, DRB1*04:270, DRB1*04:271, DRB1*04:272, DRB1*04:28, 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DRB1*15:01:06, DRB1*15:01:07, DRB1*15:01:08, DRB1*15:01:09, DRB1*15:01:10, DRB1*15:01:11, DRB1*15:01:12, DRB1*15:01:13, DRB1*15:01:14, DRB1*15:01:15, DRB1*15:01:16, DRB1*15:01:17, DRB1*15:01:18, DRB1*15:01:19, DRB1*15:01:20, DRB1*15:01:21, DRB1*15:01:22, DRB1*15:01:23, DRB1*15:01:24, DRB1*15:01:25, DRB1*15:01:26, DRB1*15:01:27, DRB1*15:01:28, DRB1*15:01:29, DRB1*15:01:30, DRB1*15:01:31, DRB1*15:01:32, DRB1*15:01:33, DRB1*15:01:34, DRB1*15:01:35, DRB1*15:01:36, DRB1*15:01:37, DRB1*15:01:38, DRB1*15:01:39, DRB1*15:01:40, DRB1*15:01:41, DRB1*15:02:01:01, DRB1*15:02:01:02, DRB1*15:02:01:03, DRB1*15:02:02, DRB1*15:02:03, DRB1*15:02:04, DRB1*15:02:05, DRB1*15:02:06, DRB1*15:02:07, DRB1*15:02:08, DRB1*15:02:09, DRB1*15:02:10, DRB1*15:02:11, DRB1*15:02:12, DRB1*15:02:13, DRB1*15:02:14, DRB1*15:02:15, DRB1*15:02:16, DRB1*15:02:17, DRB1*15:02:18, DRB1*15:02:19, DRB1*15:03:01:01, DRB1*15:03:01:02, DRB1*15:03:01:03, DRB1*15:03:02, DRB1*15:03:03, DRB1*15:03:04, DRB1*15:04, DRB1*15:05, DRB1*15:06:01, DRB1*15:06:02, DRB1*15:06:03, DRB1*15:06:04, DRB1*15:07:01, DRB1*15:07:02, DRB1*15:07:03, DRB1*15:08, DRB1*15:09, DRB1*15:10, DRB1*15:100, DRB1*15:101, DRB1*15:102, DRB1*15:103, DRB1*15:104:01, DRB1*15:104:02, DRB1*15:104:03, DRB1*15:105:01, DRB1*15:105:02, DRB1*15:106, DRB1*15:107, DRB1*15:108, DRB1*15:109, DRB1*15:110, DRB1*15:111, DRB1*15:112, DRB1*15:113N, DRB1*15:114, DRB1*15:115N, DRB1*15:116, DRB1*15:117, DRB1*15:118, DRB1*15:119, DRB1*15:11:01, DRB1*15:11:02, DRB1*15:12, DRB1*15:120, DRB1*15:121, DRB1*15:122, DRB1*15:123, DRB1*15:124, DRB1*15:125, DRB1*15:126, DRB1*15:127, DRB1*15:128, DRB1*15:129N, DRB1*15:13, DRB1*15:130, DRB1*15:131, DRB1*15:132, DRB1*15:133, DRB1*15:134N, DRB1*15:135, DRB1*15:136, DRB1*15:137N, DRB1*15:138N, DRB1*15:139, DRB1*15:14, DRB1*15:140, DRB1*15:141, DRB1*15:142, DRB1*15:143, DRB1*15:144, DRB1*15:145, DRB1*15:146, DRB1*15:147, DRB1*15:148N, DRB1*15:149, DRB1*15:150, DRB1*15:151, DRB1*15:152, DRB1*15:153, DRB1*15:154N, DRB1*15:155, DRB1*15:156, DRB1*15:157, DRB1*15:158, DRB1*15:159N, DRB1*15:15:01, DRB1*15:15:02, DRB1*15:15:03, DRB1*15:16, DRB1*15:160, DRB1*15:161, DRB1*15:162, DRB1*15:163N, DRB1*15:164Q, DRB1*15:165, DRB1*15:166, DRB1*15:167, DRB1*15:168, DRB1*15:169, DRB1*15:170, DRB1*15:17N, DRB1*15:18, DRB1*15:19, DRB1*15:20, DRB1*15:21, DRB1*15:22, DRB1*15:23, DRB1*15:24, DRB1*15:25, DRB1*15:26, DRB1*15:27, DRB1*15:28, DRB1*15:29, DRB1*15:30, DRB1*15:31:01, DRB1*15:31:02, DRB1*15:32, DRB1*15:33, DRB1*15:34, DRB1*15:35, DRB1*15:36, DRB1*15:37:01, DRB1*15:37:02, DRB1*15:38, DRB1*15:39, DRB1*15:40, DRB1*15:41, DRB1*15:42, DRB1*15:43, DRB1*15:44, DRB1*15:45, DRB1*15:46, DRB1*15:47, DRB1*15:48, DRB1*15:49, DRB1*15:50N, DRB1*15:51, DRB1*15:52, DRB1*15:53, DRB1*15:54, DRB1*15:55, DRB1*15:56, DRB1*15:57, DRB1*15:58, DRB1*15:59, DRB1*15:60, DRB1*15:61, DRB1*15:62, DRB1*15:63, DRB1*15:64, DRB1*15:65, DRB1*15:66:01, DRB1*15:66:02, DRB1*15:67, DRB1*15:68, DRB1*15:69, DRB1*15:70, DRB1*15:71, DRB1*15:72, DRB1*15:73, DRB1*15:74, DRB1*15:75, DRB1*15:76, DRB1*15:77, DRB1*15:78, DRB1*15:79, DRB1*15:80N, DRB1*15:81, DRB1*15:82, DRB1*15:83, DRB1*15:84, DRB1*15:85, DRB1*15:86, DRB1*15:87, DRB1*15:88, DRB1*15:89, DRB1*15:90, DRB1*15:91, DRB1*15:92, DRB1*15:93, DRB1*15:94, DRB1*15:95, DRB1*15:96, DRB1*15:97, DRB1*15:98, DRB1*15:99, DRB1*16:01:01, DRB1*16:01:02, DRB1*16:01:03, DRB1*16:01:04, DRB1*16:01:05, DRB1*16:01:06, DRB1*16:01:07, DRB1*16:01:08, DRB1*16:01:09, DRB1*16:01:10, DRB1*16:01:11, DRB1*16:01:12, DRB1*16:01:13, DRB1*16:01:14, DRB1*16:01:15, DRB1*16:01:16, DRB1*16:02:01:01, DRB1*16:02:01:02, DRB1*16:02:01:03, DRB1*16:02:02, DRB1*16:02:03, DRB1*16:02:04, DRB1*16:02:05, DRB1*16:02:06, DRB1*16:02:07, DRB1*16:02:08, DRB1*16:03, DRB1*16:04:01, DRB1*16:04:02, DRB1*16:05:01, DRB1*16:05:02, DRB1*16:07, DRB1*16:08, DRB1*16:09:01, DRB1*16:09:02, DRB1*16:10:01, DRB1*16:10:02, DRB1*16:11, DRB1*16:12, DRB1*16:13N, DRB1*16:14, DRB1*16:15, DRB1*16:16, DRB1*16:17, DRB1*16:18, DRB1*16:19, DRB1*16:20, DRB1*16:21N, DRB1*16:22, DRB1*16:23, DRB1*16:24, DRB1*16:25, DRB1*16:26, DRB1*16:27, DRB1*16:28, DRB1*16:29, DRB1*16:30, DRB1*16:31, DRB1*16:32, DRB1*16:33, DRB1*16:34, DRB1*16:35, DRB1*16:36, DRB1*16:37, DRB1*16:38:01, DRB1*16:38:02, DRB1*16:39, DRB1*16:40, DRB1*16:41N, DRB1*16:42, DRB1*16:43, DRB1*16:44, DRB1*16:45, DRB1*16:46, DRB1*16:47, DRB1*16:48, DRB1*16:49, DRB1*16:50, DRB1*16:51, DRB1*16:52, DRB1*16:53, DRB1*16:54, DRB1*16:55N, DRB1*16:56, and any combination thereof.

In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 21, wherein the DR beta chain comprises (i) a tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 19, (ii) a methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19, (iii) a histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19; and (iv) an isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19. In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 21, wherein the DR beta chain comprises (i) a tryptophan at a position corresponding to amino acid residue 114 of SEQ ID NO: 19, (ii) a methionine at a position corresponding to amino acid residue 143 of SEQ ID NO: 19, (iii) a histidine at a position corresponding to amino acid residue 118 of SEQ ID NO: 19; (iv) an isoleucine at a position corresponding to amino acid residue 157 of SEQ ID NO: 19; (v) a threonine at a position corresponding to amino acid residue 139 of SEQ ID NO: 19; (vi) a glutamine at a position corresponding to amino acid residue 146 of SEQ ID NO: 19; (vii) a methionine at a position corresponding to amino acid residue 163 of SEQ ID NO: 19; and (viii) a threonine at a position corresponding to amino acid residue 164 of SEQ ID NO: 19. In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence set forth in SEQ ID NO: 21.

II.A.3.b. HLA-DR Alpha Chain

In certain aspects, the DR alpha chain is selected from DRA*01:01:01:01, DRA*01:01:01:02, DRA*01:01:01:03, DRA*01:01:02, DRA*01:02:01, DRA*01:02:02, DRA*01:02:03, and any combination thereof.

In certain aspects, the MHC class II molecule comprises a DR alpha chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 24. In certain aspects, the MHC class II molecule comprises a DR alpha chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 26. In certain aspects, the MHC class II molecule comprises a DR alpha chain comprising an amino acid sequence set forth in SEQ ID NO: 24. In certain aspects, the MHC class II molecule comprises a DR alpha chain comprising an amino acid sequence set forth in SEQ ID NO: 26.

II.A.4. Signal Peptide

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

In some aspects, the signal peptide is derived from a native signal peptide. In some aspects, the signal peptide is derived from a naturally occurring DP beta chain signal peptide. In some aspects, the signal peptide comprises a naturally occurring DP beta chain signal peptide. In some aspects, the signal peptide is derived from a naturally occurring DP alpha chain signal peptide. In some aspects, the signal peptide comprises a naturally occurring DP alpha chain signal peptide.

In some aspects, the signal peptide is derived from a naturally occurring DQ beta chain signal peptide. In some aspects, the signal peptide comprises a naturally occurring DQ beta chain signal peptide. In some aspects, the signal peptide is derived from a naturally occurring DQ alpha chain signal peptide. In some aspects, the signal peptide comprises a naturally occurring DQ alpha chain signal peptide.

In some aspects, the signal peptide is derived from a naturally occurring DR beta chain signal peptide. In some aspects, the signal peptide comprises a naturally occurring DR beta chain signal peptide. In some aspects, the signal peptide is derived from a naturally occurring DR alpha chain signal peptide. In some aspects, the signal peptide comprises a naturally occurring DR alpha chain signal peptide.

In some aspects, the signal peptide is derived from a fibroin light chain (FibL) signal peptide. In some aspects, the signal peptide comprises SEQ ID NO: 9. In some aspects, the signal peptide is synthetic.

II.A.5. Transmembrane Domain

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

In some aspects, the transmembrane domain is derived from a naturally occurring DP beta chain transmembrane domain. In some aspects, the transmembrane domain comprises a naturally occurring DP beta chain transmembrane domain. In some aspects, the transmembrane domain is derived from a naturally occurring DP alpha chain transmembrane domain. In some aspects, the transmembrane domain comprises a naturally occurring DP alpha chain transmembrane domain.

In some aspects, the transmembrane domain is derived from a naturally occurring DQ beta chain transmembrane domain. In some aspects, the transmembrane domain comprises a naturally occurring DQ beta chain transmembrane domain. In some aspects, the transmembrane domain is derived from a naturally occurring DQ alpha chain transmembrane domain. In some aspects, the transmembrane domain comprises a naturally occurring DQ alpha chain transmembrane domain.

In some aspects, the transmembrane domain is derived from a naturally occurring DR beta chain transmembrane domain. In some aspects, the transmembrane domain comprises a naturally occurring DR beta chain transmembrane domain. In some aspects, the transmembrane domain is derived from a naturally occurring DR alpha chain transmembrane domain. In some aspects, the transmembrane domain comprises a naturally occurring DR alpha chain transmembrane domain.

II.A.6. Leucine Zipper

In some aspects, the beta chain and/or the alpha chain further comprises one or more leucine zipper (LZip) sequences. Any LZip sequence known in the art can be used in the compositions and methods disclosed herein. In some aspects, the beta chain and/or the alpha chain comprises an acidic LZip (αLZip), a basic LZip (βLZip), or both. In some aspects, the one or more LZip sequences are derived from a naturally occurring LZip sequence. In some aspects, the one or more LZip sequences comprise a naturally occurring LZip sequence. In some aspects, the one or more LZip sequences are synthetic. In certain aspects, the one or more LZip sequences comprise the LZip sequences set forth in SEQ ID NO: 4, 7, 14, 17, 22,or 25.

II.A.7. Linker

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

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

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

II.B. Cells

In certain aspects of the present disclosure, the MHC class II molecule used in the methods of the present disclosure is linked to or associated with a membrane of a cell. Accordingly, some aspects of the present disclosure are directed to a method of identifying an MHC class II-specific TCR comprising contacting a T cell with a cell, wherein the cell comprises a complex comprising an MHC class II molecule disclosed herein and a peptide, e.g., an epitope. In certain aspects, the beta chain of the MHC class II molecule is linked or associated with a membrane of a cell. In certain aspects, the alpha chain of the MHC class II molecule is linked or associated with a membrane of a cell. In certain aspects, the alpha chain and the beta chain of the MHC class II molecule are linked or associated with a membrane of a cell.

Any cell can be used in the methods described herein. In certain aspects the cell is a mammalian cell. In some aspects, the cell is an insect cell. In some aspects, the cell is derived from a healthy cell, e.g., a health fibroblast cell. In some aspects the cell is derived from a tumor cell. Non-limiting examples of cells that are useful in the present disclosure include K562 cells, T2 cells, HEK293 cells, HEK293T cells, A375 cells, SK-MEL-28 cells, Me275 cells, COS cells, fibroblast cells, tumor cells, or any combination thereof. In certain aspects, the cell is any cell disclosed in Hasan et al., Adv. Genet. Eng. 4(3):130 (2015), which is incorporated by reference herein in its entirety.

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

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

II.C. Soluble MHC Class II Molecules

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

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

In some aspects, the MHC class II molecule comprises a fragment of a full length MHC class II molecule, wherein one or more amino acids of the transmembrane domain of the alpha chain and/or the transmembrane domain of the beta chain are deleted. In some aspects, the MHC class II molecule comprises the extracellular domain of the alpha chain (e.g., as set forth in SEQ ID NO: 6) and/or the extracellular domain of the beta chain (e.g., as set forth in SEQ ID NO: 1 or 3). In some aspects, the MHC class II molecule comprises the extracellular domain of the alpha chain (e.g., as set forth in SEQ ID NO: 16) and/or the extracellular domain of the beta chain (e.g., as set forth in SEQ ID NO: 11 or 13). In some aspects, the MHC class II molecule comprises the extracellular domain of the alpha chain (e.g., as set forth in SEQ ID NO: 24) and/or the extracellular domain of the beta chain (e.g., as set forth in SEQ ID NO: 19 or 21).

In certain aspects, the MHC class II molecule comprises a DP alpha chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 6. In some aspects, the MHC class II molecule comprises a DP alpha chain comprising an amino acid sequence set forth in SEQ ID NO: 6.

In certain aspects, the MHC class II molecule comprises a DQ alpha chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%. at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 16. In some aspects, the MHC class II molecule comprises a DQ alpha chain comprising an amino acid sequence set forth in SEQ ID NO: 16.

In certain aspects, the MHC class II molecule comprises a DR alpha chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 24. In some aspects, the MHC class II molecule comprises a DR alpha chain comprising an amino acid sequence set forth in SEQ ID NO: 24.

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

In certain aspects, the MHC class II molecule comprises a DQ beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 11. In some aspects, the MHC class II molecule comprises a DQ beta chain comprising an amino acid sequence set forth in SEQ ID NO: 11. In certain aspects, the MHC class II molecule comprises a DQ beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 13. In some aspects, the MHC class II molecule comprises a DQ beta chain comprising an amino acid sequence set forth in SEQ ID NO: 13. In certain aspects, the MHC class II molecule comprises a DQ beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 14. In some aspects, the MHC class II molecule comprises a DQ beta chain comprising an amino acid sequence set forth in SEQ ID NO: 14. In certain aspects, the MHC class II molecule comprises a DQ beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 15. In some aspects, the MHC class II molecule comprises a DQ beta chain comprising an amino acid sequence set forth in SEQ ID NO: 15.

In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 19. In some aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence set forth in SEQ ID NO: 19. In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 21. In some aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence set forth in SEQ ID NO: 21. In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 22. In some aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence set forth in SEQ ID NO: 22. In certain aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 23. In some aspects, the MHC class II molecule comprises a DR beta chain comprising an amino acid sequence set forth in SEQ ID NO: 23.

II.D. Nucleic Acid Molecules and Vectors

Certain aspects of the present disclosure are directed to a nucleic acid molecule encoding an MHC class II molecule disclosed herein. In some aspects the nucleic acid molecule encodes an MHC class II beta chain disclosed herein. In certain aspects, the nucleic acid molecule encoding the MHC class II beta chain comprises a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with a sequence set forth in SEQ ID NO: 2, 12, or 20.

In some aspects the nucleic acid molecule encodes an MHC class II alpha chain disclosed herein. In certain aspects, the nucleic acid molecule encoding the MHC class II alpha chain comprises a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with a sequence set forth in SEQ ID NO: 7, 17, or 25.

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

In some aspects, the present disclosure is directed to a first nucleic acid molecule encoding an MHC class II beta chain disclosed herein and a second nucleic acid molecule encoding an MHC class II alpha chain disclosed herein.

Certain aspects of the present disclosure are directed to a vector or a set of vectors comprising a nucleic acid molecule disclosed herein. In some aspects, the vector is a viral vector. In some aspects, the vector is a viral particle or a virus. In some aspects, the vector is a mammalian vector. In some aspects, the vector is a bacterial vector.

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

II.E. Methods of Treating a Tumor

In certain aspects, the methods disclosed herein further comprise treating a cancer in a subject in need thereof. In some aspects, the method further comprises administering a TCR identified using the methods disclosed herein to a subject in need thereof, wherein the subject has a cancer. In some aspects, the method comprises administering a cell to the subject, wherein the cell comprises a TCR identified using the methods disclosed herein. In some aspects, the cell is a T cell.

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

In some aspects, the cancer is relapsed. In some aspects, the cancer is refractory. In some aspects, the cancer is advanced. In some aspects, the cancer is metastatic.

In some aspects, the methods disclosed herein treat a cancer in a subject. In some aspects, the methods disclosed herein reduce the severity of one or more symptom of the cancer. In some aspects, the methods disclosed herein reduce the size or number of a tumor derived from the cancer. In some aspects, the methods disclosed herein increase the overall survival of the subject, relative to a subject not provided the methods disclosed herein. In some aspects, the methods disclosed herein increase the progressive-free survival of the subject, relative to a subject not provided the methods disclosed herein. In some aspects, the methods disclosed herein lead to a partial response in the subject. In some aspects, the methods disclosed herein lead to a complete response in the subject.

Certain aspects of the present disclosure are directed to methods of treating an infection in a subject in need thereof, comprising administering to the subject an HLA class II molecule disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, or a cell disclosed herein. Non-limiting examples of infections that can be treated using the compositions and methods disclosed herein include infection by a virus (including viroids and prions), a bacterium, a fungus, a parasite, or any combination thereof. In some aspects, the virus is herpesvirus, HIV, papvavirus, measles virus, rubella virus, human papillomavirus (HPV), human T-lymphotropic virus 1, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, norovirus, and any combination thereof. In some aspects, the bacterium is selected from Streptococcus, Staphylococcus, and E. coli. In some aspects, the bacterial infection is selected from Brucellosis, Campylobacter infections, Cat-scratch disease, Cholera, Escherichia coli, Gonorrhea, Klebsiella, Enterobacter, Serratia, Legionella infections, Meningococcal infection, Pertussis, Plague, Pseudomonas infection, Salmonella infection, Shigellosis, Typhoid fever, Tularemia, Anthrax, Diphtheria, Enterococcal infection, Erysipelothricosis, Listeriosis, Nocardiosis, Pneumococcal infection, Staphylococcal infection, Streptococcal infection, and any combination thereof. In some embodiments, the parasite infection is selected from pinworm, trichomononiasis, toxoplasmosis, giardiasis, cryptosporidiosis, malaria, hookwork, ringworm, tapeworm, fluke, and any combination thereof. In some aspects, the fungal infection is selected from Candida, Malassezia furfur, dermatophytes (e.g., Epidermophyton, Microsporum, and Trichophyton), or any combination thereof.

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

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

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

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

Having generally described this disclosure, a further understanding can be obtained by reference to the examples provided herein. These examples are for purposes of illustration only and are not intended to be limiting.

EXAMPLES
Example 1—Generation of Affinity Matured HLA-DP Molecules

Cells

Peripheral mononuclear cells were obtained via density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare Life Sciences, Marlborough, Mass.). The K562 cell line is an erythroleukemic cell line with defective HLA class I/II expression. K562-based artificial APCs (aAPCs) individually expressing various HLA class II genes as a single HLA allele in conjunction with CD80 and CD83 have been reported previously (Butler et al., PloS One 7, e30229 (2012). The Jurkat 76 cell line is a T cell leukemic cell line lacking endogenous TCR, CD4, and CD8 expression. Jurkat 76/CD4 cells were generated by retrovirally transducing the human CD4 gene. A375, SK-MEL-21, SK-MEL-28, SK-MEL-37 and Me275 are melanoma cell lines. HEK293T cells and melanoma cell lines were grown in DMEM supplemented with 10% FBS and 50 μg/ml gentamicin (Thermo Fisher Scientific, Waltham, Mass.). The K562 and Jurkat 76 cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 50 μg/ml gentamicin.

Peptides

Synthetic peptides were purchased from Genscript (Piscataway, N.J.) and dissolved at 50 μg/ml in DM50. The peptide sequences are shown in Table 6.

TABLE 6

Synthetic Peptide Sequences

SEQ

ID

Name
Sequence
NO:

ABCC6_1081-1100
EVLAPVILM
50

LLNSFFNAI

ST

ABCC6_162-181
EGEISDPFR
51

FTTFYIHFA

LV

ABCC6_301-320
KALLATFGS
52

SFLISACFK

LI

ABCC6_317-336
FKLIQDLLS
53

FINPQLLSI

LI

ACRBP_450-469
GCEDVRVSG
54

WLQTEFLSF

QD

AFP_239-258
TVTKLSQKF
55

TKVNFTEIQ

KL

AIM2_18-37
TDEELDRFK
56

FFLSDEFNI

AT

AIM2_205-224
RIIIIARYY
57

RHSGFLEVN

SA

ALDH1A_143-162
RTIPIDGNF
58

FTYTRHEPI

GV

ALK_1160-1179
DELDFLMEA
59

LIISKFNHQ

NI

ANKRD30A_137-156
VYGNTALHY
60

AVYSEILSV

VA

ANXA2_241-260
LESIRKEVK
61

GDLENAFLN

LV

ARF4_73-92
RIRPLWKHY
62

FQNTQGLIF

VV

BAGE1_5-24
AVFLALSAQ
63

LLQARLMKE

ES

BAX_134-143
RTIMGWTLD
64

FLRERLLGW

IQ

BCL2L1_13-32
LSYKLSQKG
65

YSWSQFSDV

EE

BIRC5_87-106
LSVKKQFEE
66

LTLGEFLKL

DR

BIRC7_139-158
DPWTEHAKW
67

FPSCQFLLR

SK

BST2_154-173
YPSSQDSSS
68

AAAPQLLIV

LL

CA9_331-350
LTTPPCAQG
69

VIWTVFNQT

VM

CALCA_1-20
MGFQKFSPF
70

LALSILVLL

QA

CCDC110_58-77
VLQQQLESF
71

QALRMQTLQ

NV

CCNA1_366-385
KYVAELSLL
72

EADPFLKYL

PS

CCNA1_438-457
QQAIREKYK
73

ASKYLCVSL

ME

CCND1_219-238
SPNNFLSYY
74

RLTRFLSRV

IK

CD274_1-20
MRIFAVFIF
75

MTYWHLLNA

FT

CD45_1012-1031
PSKYINASF
76

IMSYWKPEV

MI

CD45_1036-1055
PLKETIGDF
77

WQMIFQRKV

KV

CD45_1204-1223
KARPGMVST
78

FEQYQFLYD

VI

CDH3_210-229
DHKPKFTQD
79

TFRGSVLEG

VL

CDKN1A_52-71
DFVTETPLE
80

GDFAWERVR

GL

CEA_266-285
PAQYSWFVN
81

GTFQQSTQE

LF

CEL_532-551
RSLRTNFLR
82

YWTLTYLAL

PT

CLCA2_132-141
CGKEGKYIH
83

FTPNFLLND

NL

CNTN2_478-497
ISRSDEGKY
84

TCFAENFMG

KA

COTL1_50-69
QQCTDDVRL
85

FAFVRFTTG

DA

CPSF1_202-221
NIIDLQFLH
86

GYYEPTLLI

LF

CPSF1_476-495
ANAAVGEPA
87

FLSEEFQNS

PE

CSAG2_11-30
GVKRKDQGF
88

LEKEFYHKT

NI

CSF1₁₃₀₁₄₉
HDKACVRTF
89

YETPLQLLE

KV

CSPG4_1741-1760
QRSEHDVLF
90

QVTQFPSRG

QL

CSPG4_2003-2022
FQIDQGEVV
91

FAFTNFSSS

HD

CSPG4_2005-2024
IDQGEVVFA
92

FTNFSSSHD

HF

CT83_9-28
SSILCALIV
93

FWKYRRFQR

NT

CTSG_44-63
AGQSRCGGF
94

LVREDFVLT

AA

CYP1B1_262-281
EQLNRNFSN
95

FILDKFLRH

CE

CYP1B1_336-355
STALQWLLL
96

LFTRYPDVQ

TR

CYP1B1_9-28
DPWPLNPLS
97

IQQTTLLLL

LS

DCT_174-193
PQFANCSVY
98

DFFVWLHYY

SV

DCT_177-196
ANCSVYDFF
99

VWLHYYSVR

DT

DCT_332-351
QKFDNPPFF
100

QNSTFSFRN

AL

DDX43_370-389
IATPGRLND
101

LQMSNFVNL

KN

DKK1_195-214
CASGLCCAR
102

HFWSKICKP

VL

EGLN3_85-104
EEGCEAISF
103

LLSLIDRLV

LY

ENAH_92-111
GSKEDANVF
104

ASAMMHALE

VL

EPCAM_172-191
TRYQLDPKF
105

ITSILYENN

VI

EPHA2_125-144
ESDLDYGTN
106

FQKRLFTKI

DT

EPHA3_126-145
ESDDDHGVK
107

FREHQFTKI

DT

EPOR_416-435
PEGASAASF
108

EYTILDPSS

QL

ERBB2_992-1011
EDLGPASPL
109

DSTFYRSLL

ED

ERBB2_993-1012
DLGPASPLD
110

STFYRSLLE

DD

EXOSC5_225-234
AASQHVFRF
111

YRESLQRRY

SK

EZH2_282-301
HTLFCRRCF
112

KYDCFLHPF

HA

FGF5_1-19
MSLSFLLLL
113

FFSHLILSA

WA

FOLHL_555-574
VYETYELVE
114

KFYDPMFKY

HL

GAGE1_117-136
VAQTGILWL
115

LMNNCFLNL

SP

GAGE3_1-20
MNLSRGKST
116

YYWPRPRRY

VQ

gp100_621-640
MKQDFSVPQ
117

LPHSSSHWL

RL

GPC3_135-154
PSLTPQAFE
118

FVGEFFTDV

SL

GPR143_120-139
AMWIQLLYS
119

ACFWWLFCY

AV

HLA-
TGPIRNGDW
120

DOB_171-190
TFQTVVMLE

MT

HPN_387-406
PGVYTKVSD
121

FREWIFQAI

KT

HSD17B12_17-36
GTVAYLALR
122

ISYSLFTAL

RV

HSD17B12_225-244
VFVQSVLPY
123

FVATKLAKI

RK

Hsp70_289-308
EGIDFYTSI
124

TRARFEELC

SD

IDO1_403-422
SFRDGDCSK
125

GFFLVSLLV

EI

IL13RA2_217-236
SSENKPIRS
126

SYFTFQLQN

IV

KAAG1_3-22
DDAAPRVEG
127

VPVAVHKHA

LH

KDM5B_381-400
TFGEMADAF
128

KSDYFNMPV

HM

KDR_765-784
IIILVGTAV
129

IAMFFWLLL

VI

KIF20A_298-317
RFSIWISFF
130

EIYNELLYD

LL

KIF2C_386-405
NQPCYRKLG
131

LEVYVTFFE

IY

KLK4_102-121
SVRHPEYNR
132

PLLANDLML

IK

LGALS3BP_374-393
QKKTLQALE
133

FHTVPFQLL

AR

LGALS9_112-131
VMVNGILFV
134

QYFHRVPFH

RV

LGSN_187-206
IAKRQLSHL
135

QASGFSLLS

AF

LGSN_287-308
TGVKEVARK
136

YNYIASFFI

ET

LGSN_296-315
YNYIASFFI
137

ETGFCDSGI

LS

LGSN_78-97
QAMAKNRLQ
138

FVRFEATDL

HG

LIMS1_34-53
QCFVCAQCF
139

QQFPEGLFY

EF

LY6K_99-118
EKRFLLEEP
140

MPFFYLKCC

KI

MAGE-
DEKVTDLVQ
141

A10_135-154
FLLFKYQMK

EP

MAGE-
ALSRKMAEL
142

A12_105-127
VHFLLLKYR

AR

MAGE-
PRKLLTQDL
143

A1_235-254
VQEKYLEYR

QV

MAGE-
AISRKMVEL
144

A2_108-127
VHFLLLKYR

AR

MAGE-
RKMVELVHF
145

A2_111-130
LLLKYRARE

PV

MAGE-
YEFLWGPRA
146

A4_270-289
LAETSYVKV

LE

MAGE-
LGSVVGNWQ
147

A6_136-155
YFFPVIFSK

AS

MAGE-
PKKLLTQYF
148

A6_242-261
VQENYLEYR

QV

MAGE-
YFPVIFGKA
149

A9_145-164
SEFMQVIFG

TD

MAGE-
PRKFITQDL
150

B1_241-260
VQEKYLKYE

QV

MAGE-
PWKLITKDL
151

B2_244-263
VQEKYLEYK

QV

MAGE-
QSPLQNPAS
152

C1_125-144
SFFSSALLS

IF

MAGE-
QSPLQIPVS
153

C1_195-214
RSFSSTLLS

IF

MAGE-
SPLQIPGSP
154

C1_371-390
SFSSTLLSL

FQ

MAGE-
SPLQIPMTS
155

C1_406-425
SFSSTLLSI

LQ

MAGEC2_373-392
PLSSCCSSF
156

SWSSFSEES

SS

MART1_32-51
ILTVILGVL
157

LLIGCWYCR

RR

MC1R_139-158
AVDRYISIF
158

YALRYHSIV

TL

MC1R_245-264
ILLGIFFLC
159

WGPFFLHLT

LI

MDK_1-20
MQHRGFLLL
160

TLLALLALT

SA

MDM2_47-66
TYTMKEVLF
161

YLGQYIMTK

RL

MET_1334-1353
VSRISAIFS
162

TFIGEHYVH

VN

MET_359-378
RSAMCAFPI
163

KYVNDFFNK

IV

MGAT5_13-32
KLGFFLVTF
164

GFIWGMMLL

HF

MMP2_479-498
AQIRGEIFF
165

FKDRFIWRT

VT

MMP2_526-545
APQEEKAVF
166

FAGNEYWIY

SA

MMP2_575-594
SKNKKTYIF
167

AGDKFWRYN

EV

MMP2_623-642
LQGGGHSYF
168

FKGAYYLKL

EN

MMP7_29-48
ELQWEQAQD
169

YLKRFYLYD

SE

MOK_156-175
QPYTEYIST
170

RWYRAPECL

LT

MPO_654-673
KGRVGPLLA
171

CIIGTQFRK

LR

MSH3_1042-1061
DPGAAEQVP
172

DFVTFLYQI

TR

MSLN_335-354
QMDRVNAIP
173

FTYEQLDVL

KH

MUC1_1035-1054
QLSTGVSFF
174

FLSFHISNL

QF

MUC16_10147-10166
SMPANFETT
175

GFEAEPFSH

LT

MUC16_10323-10342
SLPSSTPVP
176

FSSSTFTTT

DS

MUC16_11988-12007
AKTTTTFNT
177

LAGSLFTPL

TT

MUC16_2944-2963
STKAISASS
178

FQSTGFTET

PE

MUC2_94-113
ILLTIKDDT
179

IYLTRHLAV

LN

MUC5AC_4922-4941
SGWGDPHYI
180

TFDGTYYTF

LD

Nuf2_50-69
MRALQIVYG
181

IRLEHFYMM

PV

OR51E2_204-223
LVMGVDVMF
182

ISLSYFLII

RT

p53_7-26
DPSVEPPLS
183

QETFSDLWK

LL

PAK2_344-363
QIAAVCREC
184

LQALEFLHA

NQ

PAK2_485-504
VEKRGSAKE
185

LLQHPFLKL

AK

PAPOLA_121-140
PRHVDRSDF
186

FTSFYDKLK

LQ

PASD1_258-277
MFVDSDSTY
187

CSSTVFLDT

MP

PAX3_217-236
RKQRRSRTT
188

FTAEQLEEL

ER

PAX5_332-351
LTGMVPGSE
189

FSGSPYSHP

QY

PGK1_335-354
VWNGPVGVF
190

EWEAFARGT

KA

PLAC1_180-199
QAGAQEAQP
191

LQPSHFLDI

SE

PLIN2_18-37
NLPLVSSTY
192

DLMSSAYLS

TK

POTEE_952-971
NERFRCPEA
193

LFQPCFLGM

ES

PPIB_79-98
LATGEKGFG
194

YKNSKFHRV

IK

PRAME_294-313
SLQCLQALY
195

VDSLFFLRG

RL

PRDM1_659-678
YQCKVCPAK
196

FTQFVHLKL

HK

PSA_283-302
EVAAKTLPF
197

YKDYFNVPY

PL

PSA_580-599
KLNLGTVGF
198

YRTQYSSAM

LE

PSA_667-686
SHTDFYEEI
199

QEFVKDVFS

PI

PSA_837-856
ELYNRYQGG
200

FLISRLIKL

SV

PTTG1IP_81-100
CKLSSARWG
201

VCWVNFEAL

II

PXDN_1226-1245
SRLGPTLMC
202

LLSTQFKRL

RD

RAB38_131-150
QGKDVLMNN
203

GLKMDQFCK

EH

RCVRN_20-39
NTKFSEEEL
204

CSWYQSFLK

DC

RGS5_162-181
MEKDSLPRF
205

VRSEFYQEL

IK

RGS5_73-92
YGLASFKSF
206

LKSEFSEEN

LE

RhoC_16-35
CGKTCLLIV
207

FSKDQFPEV

YV

RNF43_195-214
PDYDVWILM
208

TVVGTIFVI

IL

RPS2_250-269
YLTPDLWKE
209

TVFTKSPYQ

EF

SAGE1_857-876
KVKRQFVEF
210

TIKEAARFK

KV

SART1_227-246
MDQEFGVST
211

LVEEEFGQR

RQ

SART3_135-154
RQKMSEIFP
212

LTEELWLEW

LH

SCGB2A2_74-93
LSNVEVFMV
213

ISFSSYKLF

KS

SCRN1_257-256
TLRDKASGV
214

CIDSEFFLT

TA

SDC1_262-281
VGLIFAVCL
215

VGFMLYRMK

KK

SIM2_147-166
HHHLLQEYE
216

IERSFFLRM

KC

SLAMF7_232-251
LLVPLLLSL
217

FVLGLFLWF

LK

SLC45A3_2-21
VQRLWVSRL
218

LRHRKAQLL

LV

SOX10_376-395
SQIAYTSLS
219

LPHYGSAFP

SI

SOX4_413-432
NFESMSLGS
220

FSSSSALDR

DL

SPA17_29-48
REQPDNIPA
221

FAAAYFESL

LE

SSX2_23-42
KAFDDIAKY
222

FSKEEWEKM

KA

SSX4_23-42
KAFDDIAKY
223

FSKKEWEKM

KS

STEAP1_263-282
LLGTIHALI
224

FAWNKWIDI

KQ

STEAP1_296-315
FLPIVVLIF
225

KSILFLPCL

RK

STEAP1_74-93
PIKIAAIIA
226

SLTFLYTLL

RE

STEAP1_76-95
KIAAIIASL
227

TFLYTLLRE

VI

STEAP3_218-237
LALGLFVCF
228

YAYNFVRDV

LQ

TCL1_10-29
AVTDHPDRL
229

WAWEKFVYL

DE

TERT_557-576
LRSFFYVTE
230

TTFQKNRLF

FY

TERT_558-577
RSFFYVTET
231

TFQKNRLFF

YR

TM4SF1_122-141
LDSLGQWNY
232

TFASTEGQY

LL

TPBG_241-260
LSNNSLVSL
233

TYVSFRNLT

HL

TRPC1_371-390
APKSQFGRI
234

IHTPFMKFI

IH

TRPC1_388-407
IIHGASYFT
235

FLLLLNLYS

LV

TRPC1_456-475
NQLSFVMNS
236

LYLATFALK

VV

TRPC1_578-597
QQSNDTFHS
237

FIGTCFALF

WY

TYMS_122-141
PLLTTKRVF
238

WKGVLEELL

WF

TYR_383-402
DPIFLLHHA
239

FVDSIFEQW

LR

UBXN11_258-277
QRCLRDILD
240

GFFPSELQR

LY

VENTXP1_14-33
LAAASGQNR
241

MTQGQHFLQ

KV

WDR46_273-292
RRCDRVTRL
242

EFLPFHFLL

AT

XAGE1A_33-52
CATWKVICK
243

SCISQTPGI

NL

XBP1_196-215
LQIQSLISC
244

WAFWTTWTQ

SC

ZBTB7A_99-118
VSTANVGDI
245

LSAARLLEI

PA

CLIP
LPKPPKPVS
246

KMRMATPLL

MQALPM

MAGE-
KKLLTQHFV
247

A3_243-258
QENYLEY

WT1_328-348
PGCNKRYFK
248

LSHLQMHSR

KHT

NY-
SLLMWITQC
249

ESO-
FLPVF

1_157-17

NY-
YLAMPFATP
250

ESO-
MEAELARRS

1_91-110
LA

Influenza
PKYVKQNTL
251

HA_306-318
KLAT

HIV
FRDYVDRFY
252

Gag_293-312
KTLRAEQAS

QE

DDX3Y_171-190
TGSNCPPHI
253

ENFSDIDMG

EI

Bet V
ETLLRAVES
254

1_142-153
YLL

Influenza
RGYFKMRTG
255

HA_255-270
KSSIMRS

Genes Novel TCR genes were cloned via 5′-rapid amplification of cDNA ends (RACE) PCR using SMARTer RACE 5′/3′ Kit (Takara Bio, Shiga, Japan) and sequenced as previously described. All genes were cloned into the pMX retroviral vector and transduced into cell lines using the 293GPG and PG13 cell-based retrovirus system.

Antibodies

The following antibodies were used for flow cytometry analysis: PE-conjugated anti-class 11 (9-49 (13)), APC-Cy7-conjugated anti-CD4 (RPA-T4, Biolegend, San Diego, Calif.)⁴⁴, FITC-conjugated anti-NGFR (ME20.4, Biolegend, San Diego, Calif.), PE-conjugated anti-His tag (AD1.1.10, Abcam, Cambridge, Mass.), and FITC-conjugated anti-Vβ22 (IMMU 546, Beckman Coulter, Brea, Calif.). Biotinylated DP4/NY-ESO1_157-170and DP4/WT1_329-348monomers were multimerized using PE-conjugated streptavidin (Thermo Fisher Scientific, Waltham, Mass.) according to the manufacturer's instructions. Dead cells were distinguished with the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit 465 (Thermo Fisher Scientific, Waltham, Mass.). Stained cells were analyzed with Canto II or LSRFortessa X-20 (BD Biosciences, Franklin Lakes, N.J.). Cell sorting was conducted using a FACS Aria II (BD Biosciences, Franklin Lakes, N.J.). Data analysis was performed using FlowJo software (Tree Star, Ashland, Oreg.).

The following antibodies were used for immunoblot analysis: anti-β-actin (C4, Santa Cruz Biotechnology, Santa Cruz, Calif.), rabbit polyclonal anti-MAGE-A2 (Abcam, Cambridge, Mass.), anti-CCND1 (EPR2241, Abcam, Cambridge, Mass.), HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Promega, Fitchburg, Wis.), and HRP-conjugated anti-rabbit IgG (H+L) secondary antibody (Promega, Fitchburg, Wis.).

TCR Transduction into Primary T Cells

CD3⁺ and CD4⁺ T cells were purified using the Pan T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD4⁺ T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively. Purified T cells were stimulated with aAPC/mOKT3 irradiated with 200 Gy at an E:T ratio of 20:1. Starting the following day, activated T cells were retrovirally transduced with the cloned TCR genes via centrifugation for 1 hour at 1,000×g at 32° C. for 3 consecutive days or using a Retronectin-coated plate (Takara Bio, Shiga, Japan). On the following day, 100 IU/ml IL-2 and 10 ng/ml IL-15 were added to the TCR-transduced T cells. The culture medium was replenished every 2-3 days.

Staining with Soluble CD4

The soluble CD4 (sCD4) gene was generated by fusing the human CD4 extracellular domain with a 6×His tag via a GS linker. HEK293T cells were retrovirally transduced with the sCD4 gene, and the culture supernatant containing the sCD4 monomer was harvested. sCD4 was dimerized with a PE-labeled anti-6×His tag mAb (AD1.1.10, Abcam, Cambridge, Mass.) and used. HLA class II-expressing K562 cells were stained with dimerized sCD4 in the presence of goat serum for 30 min at room temperature. The surface HLA class II expression in K562-derived cells individually expressing various class II genes was as demonstrated in FIGS. 13A-13Q.

Construction and Screening of a Multisite-Directed DPB1*04:01 Mutant cDNA Library

Multisite-directed random mutations were inserted into the DPB1*04:01 cDNA by using PCR and the following primer sets: forward: 5′-CACCACAACNNNCTTNNNTGCCACGTG-3′ (SEQ ID NO: 30) and reverse: 5′-CACGTGGCANNNAAGNNNGTTGTGGTG-3′ (SEQ ID NO: 31) for L112 and V114; forward: 5′- ACAGCTGGGGTCNNNTCCACCAACCTG-3′ (SEQ ID NO: 32) and reverse: 5′-CAGGTTGGTGGANNNGACCCCAGCTGT-3′ (SEQ ID NO: 33) for V141; forward: 5′-CAGATCNNNGTGNNNCTGGAAATGACC-3′ (SEQ ID NO: 34) and reverse: 5′-GGTCATTTCCAGNNNCACNNNGATCTG-3′ (SEQ ID NO: 35) for L156 and M158. N stands for any nucleotide. The resultant PCR fragments were fused to each other to construct a mutant full-length DPB1*04:01 cDNA expression library carrying random mutations at the positions L112, V114, V141, L156, and M158. K562 cells stably expressing the DPA1*01:03 gene were infected with recombinant retroviruses produced using the packaging cell line 293GPG at a transduction efficiency of less than 30%. The infected K562 cells were stained with soluble CD4 dimer, and the dimer-positive cells were collected using a flow cytometry cell sorter. The mutant DPB1*04:01 gene was cloned from the collected cells and retrovirally transduced into K562 cells along with the wild-type DPA1*01:03 gene as described above.

Generation of the HLA Class H Monomer and Dimer

The extracellular domain of the wild-type class II α gene was fused with an acidic leucine zipper via a GGGS linker followed by a 6×His tag via a GS linker (see SEQ ID NO: 8). The ectodomain of the class II β gene carrying mutations (see SEQ ID NO: 3) was similarly linked with a basic leucine zipper via a GGGS linker (see SEQ ID NO: 4). HEK293T cells were transfected with the α and β genes using the 293GPG cell-based retrovirus system and cultured in DMEM supplemented with 10% FBS and 50 μg/ml gentamicin. For DP4 dimer staining, HEK293 T cells stably secreting soluble DP4^L112W/V141Mprotein were grown until confluent, and the medium was changed to serum-free 293 SFM II medium (Thermo Fisher Scientific, Waltham, Mass.). After forty-eight hours, the conditioned medium was harvested and concentrated using Amicon Ultra filters (molecular weight cut-off (MWCO) 10 kDa) (MilliporeSigma, Burlington, Mass.). The soluble HLA class II-containing supernatant was then mixed with 100 μg/ml peptide of interest for 20-24 hours at 37° C. for in vitro peptide exchange. Monomer that was not subjected to peptide exchange was used as a control. The concentration of the monomer was measured by specific ELISA using a nickel-coated plate (XPressBio, Frederick, Md.) and an anti-His tag biotinylated mAb (AD1.1.10, R&D Systems, Minneapolis, Minn.). Soluble HLA class II monomer was dimerized using PE-conjugated anti-His mAb (AD1.1.10, Abcam, Cambridge, Mass.) at a 2:1 molar ratio for 1.5 hours at 4° C. for staining.

Stimulation of DP4-Restricted Antigen-Specific CD4⁺T Cells

CD4⁺ T cells were purified using a CD4⁺ T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified T cells were stimulated with DP4-expressing aAPCs pulsed with DP4-restricted peptides at 10 μg/ml and irradiated at 200 Gy at an E:T ratio of 20:1. After forty-eight hours, 10 IU/ml IL-2 and 10 ng/ml IL-15 were added to the CD4⁺ T cells. The culture medium supplemented with IL-2 (10 IU/ml) and IL-15 (10 ng/ml) was replenished every 2-3 days. After 2 weeks of stimulation, the T cells were subjected to DP4^L112W/V141Mdimer staining.

HLA Class II Dimer and Tetramer Staining

Primary T cells and Jurkat 76/CD4 T cells transduced with exogenous TCR gene were pretreated with 50 nM dasatinib (LC Laboratories, Woburn, Mass.) for 30 min at 37° C. and stained with 5-15 μg/ml class II dimer for 4-5 hours at room temperature. After washing, cell surface molecules were counterstained with an APC-Cy7-conjugated anti-CD4 mAb, a FITC-conjugated anti-NGFR mAb, and a PE-conjugated anti-V022 mAb.

ELISPOT Assay

Cytokine ELISPOT assays were performed as previously reported (see, e.g., Yamashita et al., Nat. Commun. 8:15244 (2017); and Anczurowski et al., Sci. Rep. 8:4804 (2018)).

Immunoblotting

Immunoblot analysis was performed as previously reported (see, e.g., Yamashita et al., Nat. Commun. 8:15244 (2017); and Anczurowski et al., Sci. Rep. 8:4804 (2018)).

Protein Modeling

The HLA-DP4 and human CD4 complex model structures were predicted based on structures from PDB IDs 3S5L and 3TOE using Swiss-Model workspace for quaternary structure prediction.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software, San Diego, Calif.). Unpaired two-tailed Student's t-tests were used for two-sample comparisons. No statistical method was used to predetermine sample size. The investigators were not blinded to allocation during the experiments or outcome assessment. The experiments were not randomized.

Biolayer Interferometry Sensorgram

The extracellular domain of human CD4 (residues 26-440 of NP_000607.1) followed by a GS linker and 10× histidine (His) tag was stably expressed in the human cell line A375 (SEQ ID NOs: 262-263; Table 7). Recombinant 10× His-tagged CD4 protein was purified from the supernatant with TALON metal affinity resin (Takara Bio, Shiga, Japan). The eluted protein was concentrated using an Amicon Ultra-15 spin column (MilliporeSigma, Burlington, Mass.) with a 10 kDa MWCO. Buffer was exchanged to HBS-EP (GE Healthcare Life Sciences, Marlborough, Mass.) using 10 kDa MWCO MINI Dialyzer (Thermo Fisher Scientific, Waltham, Mass.). The purity of the recombinant CD4 protein was consistently >90%, as confirmed by SDS-PAGE.

The recombinant DP4 protein consisted of extracellular domains of DPA1*01:03, and the wild-type DPB1*04:01 or L112W/V141M mutant. DPA1*01:03 was followed by an acid leucine zipper, a GS linker and a 10× histidine tag, while wild-type and mutant DPB1 was followed by a basic leucine zipper, a GS linker, and a biotinylation sequence (GLNDIFEAQKIEWHE; SEQ ID NO: 265). Both DPA and DPB genes were stably expressed in A375-BirA cells, which were transduced with the codon-optimized BirA gene encoding a leader sequence at the 5′ end and an ER retention KDEL motif at the 3′ end. Recombinant DP4 protein was purified from the supernatant with TALON metal affinity resin (Takara Bio, Shiga, Japan). Eluted protein was concentrated using Vivaspin 500 spin column (GE Healthcare Life Sciences, Marlborough, Mass.) with a 10 kDa MWCO, and reconstituted to working volume in PBS.

Binding for wild-type DP4 and DP4^L112W/V141Mwith CD4 was measured by the Octet Red system (ForteBio, Fremont, Calif.). Experiments were performed at 25° C. using a 96-well OptiPlate (Perkin Elmer, Waltham, Mass.), with a 200-1 sample volume and constant shaking at 1,000 rpm. The biotinylated recombinant DP4 was loaded onto streptavidin-coated biosensors (ForteBio, Fremont, Calif.) until saturation, followed by baseline measurement in the HBS-EP buffer. Association was measured by incubating the loaded sensors for 400 sec with titrated concentrations of recombinant CD4 (0.8125 to 26 μM) before 300 sec dissociation in HBS-EP buffer alone. The steady-state analysis was fitted using a one-site specific binding model in GraphPad Prism 7.0

TABLE 7

Soluble 10x His-tagged CD4 Nucleic Acid Sequence

Fibroin L Signal Peptide; CD4; Gly/Ser Linker and

His tag sequences (10X))(SEQ ID NO: 262)

ATGATGCGGCCCATCGTGCTGGTGCTGCTGTTTGCCACATCTGCCCTGGC

CAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACCTGTA

CAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAG

ATAAAGATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAA

GCTGAATGATCGCGCTGACTCAAGAAGAAGCCTTTGGGACCAAGGAAACT

TTCCCCTGATCATCAAGAATCTTAAGATAGAAGACTCAGATACTTACATC

TGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAATTGCTAGTGTTCGGATT

GACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGA

CCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCA

AGGGGTAAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGA

GCTCCAGGATAGTGGCACCTGGACATGCACTGTCTTGCAGAACCAGAAGA

AGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTTTCCAGAAGGCCTCC

AGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAGTTCTCCTTCCCACT

CGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGGCAGG

CGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAAC

AAGGAAGTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGG

CAAGAAGCTCCCGCTCCACCTCACCCTGCCCCAGGCCTTGCCTCAGTATG

CTGGCTCTGGAAACCTCACCCTGGCCCTTGAAGCGAAAACAGGAAAGTTG

CATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACTCAGCTCCAGAAAAA

TTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTGAGCT

TGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGGCGGTG

TGGGTGCTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTC

GGGACAGGTCCTGCTGGAATCCAACATCAAGGTTCTGCCCACATGGGGCA

GCCACCACCACCATCACCATCATCATCACCATTGA

Fibroin L Signal Peptide; CD4; Gly/Ser Linker and

His tag sequences (10X))(SEQ ID NO: 263)

MMRPIVLVLLFATSALAKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQ

IKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYI

CEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSP

RGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKAS

SIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKN

KEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGHLTLALEAKTGKL

HQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAV

WVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWGSHHHHHHHHHH

Example 2—L112W/V141M Substitutions of the DPβ Chain Enhance the Binding of DP to CD4

A cDNA expression library was generated of the DPB1*04:01 (DP40) gene carrying random mutations at L112, V114, V141, L156, and M158, which corresponds to L114, V116, V143, L158, and M160 of the DR1β chain, respectively, and coexpressed the library along with the wild-type DPA1*01:03 (DPα) gene in class II-deficient K562 cells. After two rounds of screening using soluble CD4 protein (sCD4), cell populations with enhanced CD4 binding were isolated, from which a mutant DP4β gene carrying L112W, V114M, V141M, and M158I substitutions was molecularly cloned. When ectopically expressed in the K562 cells, the mutant DP4 molecules consisting of the wild-type DPα chain and cloned mutant DP4β chain carrying L112W, V114M, V141M, and M158I substitutions (DP4^{L112W/V114M/V141M/M158I}) indeed showed enhanced binding to sCD4 compared with the wild-type DP4 molecules, excluding the possibility that enhanced CD4 binding was an artifact of screening processes (FIGS. 1A-1F).

To determine which of the four mutations is critical for enhanced CD4 binding, a reversion mutagenesis study was conducted. All the possible reversion DP4 mutants were reconstituted on class II-negative K562 cells and stained with sCD4. Both the L112W and V141M but not V114M or M158I single substitutions individually enhanced the binding of DP4 to sCD4 (FIG. 1G). Importantly, the L112W/V141M double mutations (DP4^L112W/V141M) synergistically enhanced the DP4/CD4 binding (FIG. 1G). Interestingly, both the V114M and M158I single replacements appeared to have a negative effect on the enhanced binding enabled by the DP4^L112W/V141Mmutations (FIG. 1G). Previous studies have estimated that the K_Dvalue between CD4 and HLA class II is >2 mM. Using biolayer interferometry (BLI) binding assay, the affinity of DPR^L112/V141Mfor CD4 was measured. While no binding was detected between wild-type DP4 and CD4, DP4^L112W/V141Mbound to CD4 with a K_Dof 8.9 μM±1.1 (FIG. 1H and FIGS. 1X-1BK). This value represents an at least 200-fold improvement in the binding affinity. Further, the observed affinity between CD4 and DP4^L112W/V141Mis higher than that between human CD8 and HLA class I (˜200 μM) and is comparable to that between mouse CD8 and mouse MHC Class I (˜10 μM). To confirm that enhanced binding between DP4^L112W/V141Mand CD4 leads to an enhanced CD4⁺ T cell response, a comparison was conducted of the immunostimulatory capacity of artificial APCs (aAPCs) expressing either wild-type DP4 or DP4^L112W/V141Mas a single class II allele using DP4/WT1 TCR (clone 9)-transduced CD4⁻ and CD4⁺ Jurkat 76 T cells as responder cells. As expected, DP4^L112W/V141Mcarrying aAPCs demonstrated enhanced T cell stimulatory activity in a CD4-dependent manner (FIG. 1I).

Next other DP alleles to CD4 were analyzed to determine whether the L112W/V141M mutations also enhance binding. Although none of the wild-type DP2, DP5, or DP8 bound to CD4, all three molecules bound to CD4 strongly when the L112W/V141M double mutations were introduced in the DPβ chains of these molecules (FIGS. 1I-1W). A structural model (FIGS. 2A-2D) constructed based on a previous report revealed that in the DP4^L112W/V141M-CD4 complex, the two L112W/V141M mutations apparently induced a hydrophobic effect at the positions, K35, Q40, and T45 of CD4. These results show that L112W/V141M mutations can enhance the CD4 binding of at least all 4 of the DP alleles tested.

Example 3—Affinity-Matured DP4^L112W/V141MMultimers Specifically Stain Cognate TCRs

To determine the effect of the L112W/V141M double mutations of DP40 on DP4 multimer staining, a soluble DP4^L112W/V141Mmonomer was produced, which was then dimerized with an anti-His tag mAb. Primary T cells were individually transduced with three different DP4-restricted TCRs specific for MAGE-A3 (clone R12C9), WT1 (clone 9), and NY-ESO-1 (clone 5B8) and then stained with cognate DP4^L112W/V141Mdimers. As shown in FIGS. 3A-3P, each DP4^L112W/V141Mdimer specifically stained CD4⁺ T cells expressing the cognate TCR. Costaining of R12C9- and clone 9-transduced T cells with an anti-Vβ22 mAb and anti-NGFR mAb, respectively, along with the respective DP4^L112W/V141Mdimer confirmed that virtually all TCR-transduced CD4⁺ T cells were successfully stained with the respective DP4^L112W/V141Mdimers (FIGS. 4A-4H). Compared with conventional wild-type DP4 tetramers, our novel DP4^L112W/V141Mdimers stained both DP4/WT1 and DP4/NY-ESO-1 T cells better than conventional wild-type DP4 tetramers (FIGS. 5A-5P). Notably, the conventional wild-type DP4/NY-ESO-1 tetramer was unable to stain cognate T cells even at the highest concentration available (data not shown).

Example 4—DP4^L112W/V141MDimer Technology is Robust and Versatile

To demonstrate the robustness and versatility of the DP4^L112W/V141Mmultimer staining, a comprehensive screening was performed for the in vitro immunogenicity of potential DP4-restricted peptides derived from an array of tumor-associated antigens (Table 6). One hundred and ninety-six DP4-restricted and tumor-associated antigen-derived 20-mer peptides were predicted using a peptide prediction algorithm (NetMHC2 ver.2.2) and chemically synthesized (Table 6). The frequency of antigen-specific CD4⁺ T cells is generally very low in the periphery; therefore, primary CD4⁺ T cells isolated from six DP4⁺ melanoma patients were stimulated only once with DP4-aAPCs individually pulsed with the 196 peptides and stained with cognate DP4^L112W/V141Mdimers. To avoid potential in vitro priming, weak stimulatory conditions were utilized. As shown in FIGS. 6A-6F, 103 predicted DP4 peptides were immunogenic, at least in vitro.

To validate the dimer staining results, we cloned seven DP4-restricted TCR genes specific for CCND1_219-238, HSD17B12_225-244, LGSN_296-315, MAGE-A2_108-127, and MUC5AC_4922-4941(FIGS. 7A-7L and Table 8) from the dimer-positive T cells. When clonotypically reconstituted in human CD4⁺ TCR-deficient T cells, all these TCRs were successfully stained by the cognate DP4^L112W/V141Mdimers (FIGS. 8A-8X) and were functional in a DP4-restricted and antigen-specific manner (FIGS. 9A-9G).

Among the four TCRs individually expressed in primary T cells, three TCRs, i.e., 03-CCND1_219-238, 06-MAGE-A2_108-127, and 05-MUC5AC_4922-4941, were able to recognize cognate peptides that were endogenously processed and presented by DP4 (FIGS. 10A-10Q and 11A-11E). Importantly, 06-MAGE-A2_108-127-transduced primary T cells were able to recognize melanoma cell lines in a DP4- and MAGE-A2-dependent manner (FIGS. 12A-12E).

TABLE 8

DP4-Restricted TCRs

No.
Peptide
TRAV
TRAJ
TCR-alpha CDR 3
TRBV
TRBJ
TCR-beta CDR 3

03
CCND1_219-238
2*01
21*01
CAVCTLYNFNKFYF
6-
2-1*01
CASLTDNNEQFF

(SEQ ID NO: 36)
5*01

(SEQ ID NO: 43)

05
HSD17B12_225-244
22*01
18*01
CAVAPYDRGSTLGRLY
19*01
2-5*01
CASSTGQGLETQYF

F (SEQ ID NO: 37)

(SEQ ID NO: 44)

09
HSD17B12_225-244
27*01
33*01
CAGVKDSNYQLIW
30*01
2-1*01
CAWSSYNEQFF (SEQ

(SEQ ID NO: 38)

ID NO: 45)

05
LGSN_296-315
9-2*03
32*01
CALSDLSYGGATNKLI
27*01
1-5*01
CASSKGQGLGNQPQHF

F (SEQ ID NO: 39)

(SEQ ID NO: 46)

03
MAGE-A2_108-127
36/DV7*
40*01
CAVEVNSGTYKYIF
2*01
1-1*01
CASRRDLAAFF (SEQ

04

(SEQ ID NO: 40)

ID NO: 47)

06
MAGE-A2_108-127
19*01
40*01
CALSVGTYKYIF(SEQ
7-
2-5*01
CASSPGTGGRETQYF

ID NO: 41)
9*01

(SEQ ID NO: 48)

05
MUC5AC_4922-4941
38-1*03
58*01
CAFMKRAETSGSRLTF
6-
2-5*01
CASSYWPTRETQYF

(SEQ ID NO: 42)
2*01

(SEQ ID NO: 49)

In contrast to CD8, the role and function of CD4 as a coreceptor has yet to be fully elucidated. This lack of information exists mainly because the binding between CD4 and class II is exceptionally weak, which significantly limits research on the role of the association between CD4 and class II. In this study, an affinity-matured form of HLA-DP4, i.e., DP4^L112W/V141M, was isolated with enhanced CD4 binding, and a novel DP4^L112W/V141Mdimer technology was developed, which introduces robustness and rigorousness in the detection of DP4-restricted antigen-specific CD4⁺ T cells.

Using this DP4^L112W/V141Mdimer technology, DP4-restricted antitumor T cell responses were comprehensively studied in vitro and multiple DP4-restricted immunogenic peptides and cognate TCR genes were identified. HLA-DP4 is the most prevalent HLA allele in many ethnic groups and belongs to the DP^84Glygroup. Unlike other class II molecules, DP^84Glymolecules such as DP4 constitutively present peptides derived from endogenous sources regardless of the invariant chain and HLA-DM expression. The improved presentation of endogenous peptides via class II is correlated with improved survival of cancer patients. Notably, a first-in-human class II-restricted TCR gene therapy indeed targeted a DP4-restricted MAGE-A3 peptide (see, e.g., Yao et al., J. Immunother. 39:191-201 (2016)). The DP^84Glygenotype, such as in DP2 and DP4, acts as a risk allele for anti-neutrophil cytoplasmic autoantibody-associated vasculitis. DP4 molecules, which can constitutively present peptides derived from endogenous tumor-associated antigens, may induce more clinically relevant antitumor responses than other class II molecules, serving as a protective class II allele.

To identify affinity-matured class II molecules, the present examples detail multiple mutations in the β-chain but not the α-chain because the β-chain has a more direct interaction with CD4 than the α chain. It is possible that additional mutations of the α- and/or β-chains can further enhance the binding between class II and CD4. However, the use of such soluble class II molecules with excessive CD4 binding capabilities may cause nonspecific staining of CD4⁺ T cells, thereby having a detrimental effect.

In conclusion, CD4⁺ T cells play a critical role in the development of autoimmune diseases and protection against pathogenic infections and cancers. The novel HLA class II multimer technology described herein may better facilitate the study of HLA class II-restricted CD4⁺ T cell responses across HLA-DP alleles.

Example 5—Generation of Affinity Matured HLA-DQ Molecules

Cells

Peripheral mononuclear cells were obtained via density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare Life Sciences, Marlborough, Mass.). The K562 cell line is an erythroleukemic cell line with defective HLA class I/II expression. A375 is melanoma cell lines. HEK293T cells and A375 cells were grown in DMEM supplemented with 10% FBS and 50 μg/ml gentamicin (Thermo Fisher Scientific, Waltham, Mass.). The K562 and Jurkat 76 cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 50 μg/ml gentamicin.

Peptides

Synthetic peptides were purchased from Genscript (Piscataway, N.J.) and dissolved at 50 μg/ml in DMSO.

Antibodies

The following antibodies were used for flow cytometry analysis: PE-conjugated anti-class II (9-49 (I3), Beckman Coulter, Brea, Calif.; T039), APC-Cy7-conjugated anti-CD4 (RPA-T4, Biolegend, San Diego, Calif.) and PE-conjugated anti-His tag (AD1.1.10, Abcam, Cambridge, Mass.). Dead cells were distinguished with the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit 465 (Thermo Fisher Scientific, Waltham, Mass.). Stained cells were analyzed with Canto II or LSRFortessa X-20 (BD Biosciences, Franklin Lakes, N.J.). Cell sorting was conducted using a FACS Aria II (BD Biosciences, Franklin Lakes, N.J.). Data analysis was performed using FlowJo software (Tree Star, Ashland, Oreg.).

TCR Transduction into Primary T Cells

Staining with Soluble CD4

Generation of the HLA Class II Monomer and Dimer

The extracellular domain of the wild-type class II a gene was fused with an acidic leucine zipper via a GGGS linker followed by a 6×His tag via a GS linker (see SEQ ID NO: 18). The ectodomain of the class II β gene carrying mutations (see SEQ ID NO: 13) was similarly linked with a basic leucine zipper via a GGGS linker (see SEQ ID NO: 14). HEK293T cells and A375 cells were transfected with the α and β genes using the 293GPG cell-based retrovirus system and cultured in DMEM supplemented with 10% FBS and 50 μg/ml gentamicin. For dimer staining, A375 cells stably secreting soluble DQ5^{L114W/V143M+4reps}(which possesses the N110Q/I116V/S118H/P146N replacements (4reps) in addition to L114W/V143M) and DQ6^{L114W/V143M+3reps}(which possesses the N110Q/S118H/P146N replacements (3reps) in addition to L114W/V143M) protein were grown until confluent, and after forty-eight hours, the medium was harvested. The soluble HLA class II-containing supernatant was then mixed with 100 μg/ml peptide of interest for 20-24 hours at 37° C. for in vitro peptide exchange. Monomer that was not subjected to peptide exchange was used as a control. The concentration of the monomer was measured by specific ELISA using a nickel-coated plate (XPressBio, Frederick, Md.) and an anti-His tag biotinylated mAb (AD1.1.10, R&D Systems, Minneapolis, Minn.). Soluble HLA class II monomer was dimerized using PE-conjugated anti-His mAb (AD1.1.10, Abcam, Cambridge, Mass.) at a 2:1 molar ratio for 1.5 hours at 4° C. for staining.

HLA Class II Dimer Staining

Primary T cells transduced with exogenous TCR gene were pretreated with 50 nM dasatinib (LC Laboratories, Woburn, Mass.) for 30 min at 37° C. and stained with 5-15 μg/ml class II dimer for 4-5 hours at room temperature. After washing, cell surface molecules were counterstained with an APC-Cy7-conjugated anti-CD4 mAb.

Statistical Analysis

Example 6—DQ Molecules with Enhanced CD4 Binding Capacities

Affinity enhanced DQ molecules were generated by introducing L114W/V143M mutations, to determine if these substitutions could improve the binding of HLA-DQ molecules such as DQ5 (DQA1*01:01-DQB1*05:01) to CD4. DQB1*05:01 encodes four different amino acids at positions 110, 116, 118, and 146 in addition to 114 and 143. We therefore generated K562 cells expressing DQ5^{L114W/V143M+4reps}, which possesses the N110Q/I116V/S118H/P146N replacements (4reps) in addition to L114W/V143M (FIG. 14A), and stained the cells with sCD4. K562 cells expressing DQ5^{L114W/V143M+4reps}but not DQ5^L114W/V143M, DQ5^4reps, or wild-type DQ5 demonstrated enhanced CD4 binding (FIGS. 14B-14C). Importantly, a series of K562 cells individually expressing various DQ5^{L114W/V143M+4reps}mutants with a single amino acid reversal at one of the four positions lacked the enhanced CD4 binding capability (FIG. 14D). These results suggest that the four additional replacements at N110Q, I116V, S118H, and P146N are critical for the effectiveness of the L114W/V143M mutations in the observed enhanced DQ5:CD4 binding.

DQβ chains such as DQB1*02:01, 04:02, and 06:01 encode distinct amino acids at positions 110, 118, and 146 but not at 116 (FIG. 14E). Unlike DQB1*05:01, DQB1*02:01, 04:02, and 06:01 encode Val at position 116, similar to DPB1*04:01, which codes for Val at position 114. All the DQ2^{L114W/V143M+3reps}, DQ4^{L114W/V143M+3reps}, and DQ6^{L114W/V143M+3reps}mutants, the β chains of which carry the N110Q, S118H, and P146N replacements (3reps) along with L114W/V143M, showed enhanced CD4 binding activity (FIG. 14F).

Example 7—Affinity-Matured DQ Dimers Specifically and Robustly Stained Cognate TCRs

The ability of the affinity-matured DQ dimers carrying the mutations described in example 2 were evaluated for the ability to identify antigen-specific CD4⁺ T cells. The DQ5^{L114W/V143M+4reps}and DQ6^{L114W/V143M+3reps}dimers successfully stained the DQ5-restricted DDX3Y-specific TCR (E6) and DQ6-restricted influenza virus-specific TCR (DM2), respectively (FIGS. 15A-15B).

Example 8—Generation of Affinity Matured HLA-DR Molecules

Cells

Peptides

Synthetic peptides were purchased from Genscript (Piscataway, N.J.) and dissolved at 50 μg/ml in DMSO.

Antibodies

The following antibodies were used for flow cytometry analysis: PE-conjugated anti-class II (9-49 (I3)), APC-Cy7-conjugated anti-CD4 (RPA-T4, Biolegend, San Diego, Calif.)⁴⁴, PE-conjugated anti-His tag (AD1.1.10, Abcam, Cambridge, Mass.), and FITC-conjugated anti-V022 (IMMU 546, Beckman Coulter, Brea, Calif.). Dead cells were distinguished with the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit 465 (Thermo Fisher Scientific, Waltham, Mass.). Stained cells were analyzed with Canto II or LSRFortessa X-20 (BD Biosciences, Franklin Lakes, N.J.). Cell sorting was conducted using a FACS Aria II (BD Biosciences, Franklin Lakes, N.J.). Data analysis was performed using FlowJo software (Tree Star, Ashland, Oreg.).

TCR Transduction into Primary T Cells

CD4⁺ T cells were purified using the CD4⁺ T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified T cells were stimulated with aAPC/mOKT3 irradiated with 200 Gy at an E:T ratio of 20:1. Starting the following day, activated T cells were retrovirally transduced with the cloned TCR genes via centrifugation for 1 hour at 1,000×g at 32° C. for 3 consecutive days or using a Retronectin-coated plate (Takara Bio, Shiga, Japan). On the following day, 100 IU/ml IL-2 and 10 ng/ml IL-15 were added to the TCR-transduced T cells. The culture medium was replenished every 2-3 days.

Staining with Soluble CD4

Generation of the HLA Class H Monomer and Dimer

The extracellular domain of the wild-type class II α gene was fused with an acidic leucine zipper via a GGGS linker followed by a 6×His tag via a GS linker (see SEQ ID NO: 26). The ectodomain of the class II β gene carrying mutations (see SEQ ID NO: 21) was similarly linked with a basic leucine zipper via a GGGS linker (see SEQ ID NO: 22). HEK293T cells were transfected with the α and β genes using the 293GPG cell-based retrovirus system and cultured in DMEM supplemented with 10% FBS and 50 μg/ml gentamicin. For DR1 dimer staining, HEK293T cells stably secreting soluble DR1^{L114W/V143M+2reps}, DR7^{L114W/V143M+2reps}and DR11^{L114W/V143M+2reps}(which possesses the S118H/T157I replacements (2reps) in addition to L114W/V143M) protein were grown until confluent, and the after forty-eight hours, the medium was harvested. The soluble HLA class II-containing supernatant was then mixed with 100 μg/ml peptide of interest for 20-24 hours at 37° C. for in vitro peptide exchange. Monomer that was not subjected to peptide exchange was used as a control. The concentration of the monomer was measured by specific ELISA using a nickel-coated plate (XPressBio, Frederick, Md.) and an anti-His tag biotinylated mAb (AD1.1.10, R&D Systems, Minneapolis, Minn.). Soluble HLA class II monomer was dimerized using PE-conjugated anti-His mAb (AD1.1.10, Abcam, Cambridge, Mass.) at a 2:1 molar ratio for 1.5 hours at 4° C. for staining.

HLA Class H Dimer Staining

Primary T cells transduced with exogenous TCR gene were pretreated with 50 nM dasatinib (LC Laboratories, Woburn, Mass.) for 30 min at 37° C.⁴⁶and stained with 5-15 μg/ml class II dimer for 4-5 hours at room temperature. After washing, cell surface molecules were counterstained with an APC-Cy7-conjugated anti-CD4 mAb and a PE-conjugated anti-V022 mAb.

Protein Modeling

The HLA-DR1 and human CD4 complex model structures were predicted based on structures from PDB IDs 3S5L and 3TOE using Swiss-Model workspace for quaternary structure prediction.

Statistical Analysis

Biolayer Interferometry and Steady-State Analysis

The recombinant DR1 protein consisted of extracellular domains of DRA1*01:01, and the wild-type DRB1*01:01 or L114W/V143M+2reps mutant. DRA1*01:01 was followed by an acid leucine zipper, a GS linker and a 10×histidine tag, while wild-type and mutant DRB1 was followed by a basic leucine zipper, a GS linker, and a biotinylation sequence (GLNDIFEAQKIEWHE; SEQ ID NO: 264). Both DRA and DRB genes were stably expressed in A375-BirA cells, which were transduced with the codon-optimized BirA gene encoding a leader sequence at the 5′ end and an ER retention KDEL motif at the 3′ end. Recombinant DR1 protein was purified from the supernatant with TALON metal affinity resin (Takara Bio, Shiga, Japan). Eluted protein was concentrated using Vivaspin 500 spin column (GE Healthcare Life Sciences, Marlborough, Mass.) with a 10 kDa MWCO, and reconstituted to working volume in PBS.

Binding for wild-type DR1 and DR1^{L114W/V143M+2reps}with CD4 was measured by the Octet Red system (ForteBio, Fremont, Calif.). Experiments were performed at 25° C. using a 96-well OptiPlate (Perkin Elmer, Waltham, Mass.), with a 200-μl sample volume and constant shaking at 1,000 rpm. The biotinylated recombinant DR1 was loaded onto streptavidin-coated biosensors (ForteBio, Fremont, Calif.) until saturation, followed by baseline measurement in the HBS-EP buffer. Association was measured by incubating the loaded sensors for 400 sec with titrated concentrations of recombinant CD4 (0.8125 to 26 μM) before 300 sec dissociation in HBS-EP buffer alone. The steady-state analysis was fitted using a one-site specific binding model in GraphPad Prism 7.0.

Example 9—DR Molecules with Enhanced CD4 Binding Capacities

Affinity enhanced DR molecules were generated by introducing L114W/V143M mutations, to determine if these substitutions could improve the binding of HLA-DR molecules such as DR1 allele (DRA1*01:01-DRB1*01:01) to CD4. DRB1*01:01 encodes six different amino acids at positions 118, 139, 146, 157, 163 and 164 in addition to 114 and 143 (FIG. 17A). DR1^{L114W/V143M+6reps}, showed enhanced CD4 binding compared with DR1^L114W/V143Mand wild-type DR1 (FIGS. 17B and 17C). A library of DR1^{L114W/V143M+6reps}-derived mutants with a single amino acid reversal at either S118H or T157I but not at the 4 other positions showed decreased CD4 binding capability, suggesting that both the S118H and T157I mutations are critical (FIG. 17D).

Indeed, the CD4 binding capacity of DR1^{L114W/V143M+2reps}, which possesses the L114W/V143M+S118H/T157I replacements (2reps) in the β chain, was comparable to that of DR1^{L114W/V143M+6reps}(FIG. 17E). These results suggest that the two additional replacements at S118H and T157I are pivotal for the function of the L114W/V143M mutations in the improvement of the binding of DR1 to CD4.

DRβ chains such as DRB1*03:01, 04:01, 07:01, 10:01, 11:01, and 13:01 encode different amino acids at positions 118, 139, 146, 157, 163, and 164 in addition to 114 and 143 (FIG. 17F). Interestingly, comparison of CD4 binding activity between the DR1^{L114W/V143M+2reps}and DR1^{L114W/V143M+6reps}mutants showed that, unlike for DR1, the L114W/V143M+2reps mutations enabled improved CD4 binding compared to the L114W/V143M+6reps mutations for DR3, DR4, DR7, DR10, DR11, and DR13 (FIGS. 17G-17L).

Using a biolayer interferometry (BLI) binding assay, the affinities of wildtype DR1 and DR1^{L114W/V143M+2reps}for CD4 were measured. While no binding was detected between wildtype DR1 and CD4 (FIG. 17M), DR1^{L114W/V143M+2reps}bound to CD4 with a KD of 14 μM+2.3 (FIGS. 17N-17O).

Example 10—Affinity-Matured DR Dimers Specifically and Robustly Stained Cognate TCRs

The ability of the affinity-matured DR dimers carrying the mutations described in example 2 were evaluated for the ability to identify antigen-specific CD4⁺ T cells. The DR1^{L114W/V143M+2reps}, DR7^{L114W/V143M+2reps}, and DR11^{L114W/V143M+2reps}dimers specifically stained the DR1-restricted TCRs HA1.7 and SB95, DR7-restricted TCR SD334, and DR11-restricted TCR F24, respectively (FIGS. 18A-18C). Costaining of F24-transduced CD4⁺ T cells with an anti-Vβ22 mAb, along with the respective DR11^{L114W/V143M+2reps}dimers, confirmed that virtually all the TCR-transduced CD4⁺ T cells were successfully stained with the respective DR11^{L114W/V143M+2reps}dimers (FIG. 18D).

A structural model of the complex consisting of CD4 and DR1^{L114W/V143M+2reps}also showed a potential hydrophobic effect of the L114W/V143M replacements (FIGS. 19A-19B). Furthermore, hydrophobic stacking was observed between P96 of the α-chain and S118H of the 3-chain (FIG. 19C), and the T157I replacement was found to localize in the β-sheet surrounding Vi 19, F112, I127, V129, L147 and T157 (FIG. 19D). It is possible that additional mutations of the α- and/or β-chains can further enhance the binding between class II and CD4. However, the use of such soluble class II molecules with excessive CD4 binding capabilities may cause nonspecific staining of CD4⁺ T cells, thereby having a detrimental effect.

Example 11

DP4 multimer staining of endogenous (untransduced) antigen specific CD4⁺ T cells was analyzed. The novel DP4^L112W/V141Mdimers positively stained endogenous TRPC1_578-597-specific CD4⁺ T cells (FIGS. 21A-21B) more strongly than the conventional DP4 dextramer (FIGS. 21C-21D). The DP4^L112W/V141Mdimers showed markedly improved staining of endogenous (untransduced) NY-ESO-1_157-170specific CD4⁺ T cells (FIGS. 22A-22B; Table 9) compared with conventional tetramers (FIGS. 22C-22D) or dextramers (FIGS. 22E-22F).

TABLE 9

DP4-Restricted TCRs

Donor

No.
TRAV
TRAJ
TCR-alpha CDR3
TRBV
TRBJ
TCR-beta CDR3

HD04
8-2*01
32*02
CVVSGGVNGGATNKLIF
7-9*01
2-7*01
CASSLTGGVSYEQYF

SEQ ID NO: 278

SEQ ID NO: 279

c6
13-1*01
18*01
CAASVRGSTLGRLYF
7-8*01
2-2*01
CASSLGTGGTGELFF

SEQ ID NO: 280

SEQ ID NO: 281

c12
8-4*01
18*01
CAVSGGRGSTLGRLYF
29*01
1-2*01
CSVQGGLDSNYGYTF

SEQ ID NO: 282

SEQ ID NO: 283

c17
13-1*01
18*01
CAASVRGSTLGRLYF
7-8*01
2-2*01
CASSLGTGGTGELFF

SEQ ID NO: 284

SEQ ID NO: 285

c23
13-1*01
18*01
CAASVRGSTLGRLYF
7-8*01
2-2*01
CASSLGTGGTGELFF

SEQ ID NO: 286

SEQ ID NO: 287

c26
38-2/
21*01
CAYRSNNFNKFYF
5-1*01
1-2*01
CASSLNTGAGYGYTF

DV8*01

SEQ ID NO: 288

SEQ ID NO: 289

c31
13-1*01
18*01
CAASVRGSTLGRLYF
7-8*01
2-2*01
CASSLGTGGTGELFF

SEQ ID NO: 290

SEQ ID NO: 291

c37
13-1*01
18*01
CAASVRGSTLGRLYF
7-8*01
2-2*01
CASSLGTGGTGELFF

SEQ ID NO: 292

SEQ ID NO: 293

c39
2*01
9*01
CAVEERTGGFKTIF
2*01
2-2*01
CASSLPSGGAPGTGELFF

SEQ ID NO: 294

SEQ ID NO: 295

c52
8-4*01
18*01
CAVSGGRGSTLGRLYF
29*01
1-2*01
CSVQGGLDSNYGYTF

SEQ ID NO: 296

SEQ ID NO: 297

c87
13-1*01
18*01
CAASVRGSTLGRLYF
7-8*01
2-2*01
CASSLGTGGTGELFF

SEQ ID NO: 298

SEQ ID NO: 299

c2
4*01
39*01
CLVGDLGANAGNMLTF
19*01
2-2*01
CASSIATTNTGELFF

SEQ ID NO: 300

SEQ ID NO: 301

c4
4*01
39*01
CLVGDLGANAGNMLTF
11-3*01
2-7*01
CASSLETGTNYEQYF

SEQ ID NO: 302

SEQ ID NO: 303

c6
8-3*01
32*02
CAVALYGGATNKLIF
7-9*3
2-1*01
CASSLDIGNNEQFF

SEQ ID NO: 304

SEQ ID NO: 305

c9
25*01
53*01
CAGRSGGSNYKLTF
19*01
2-2*01
CASSIATTNTGELFF

SEQ ID NO: 306

SEQ ID NO: 307

c29
25*01
53*01
CAGRSGGSNYKLTF
19*01
2-2*01
CASSIATTNTGELFF

SEQ ID NO: 308

SEQ ID NO: 309

c30
25*01
53*01
CAGRSGGSNYKLTF
19*01
2-2*01
CASSIATTNTGELFF

SEQ ID NO: 310

SEQ ID NO: 311

c32
25*01
53*01
CAGRSGGSNYKLTF
19*01
2-2*01
CASSIATTNTGELFF

SEQ ID NO: 312

SEQ ID NO: 313

c33
25*01
53*01
CAGRSGGSNYKLTF
19*01
2-2*01
CASSIATTNTGELFF

SEQ ID NO: 314

SEQ ID NO: 315

Next, ex vivo staining was performed of memory CD4⁺ T cells with DP4^L112W/V141Mdimers specific to a series of pathogen-associated peptides without in vitro stimulation. A small subset of the CD4⁺ T cells were positively stained with DP4^L112W/V141Mdimers for tetanus toxin_948-968(TT_948-968), herpes simplex virus type-2-UL21_283-302(HSV-2-UL21_283-302), and respiratory syncytial virus glycoprotein_162-175(RSV-GP_162-175) (FIGS. 23A-23Y). Next, we established endogenous (untransduced) single-cell clones by limiting dilution from RSV-GP_162-175(FIGS. 24A-24V) and TT_948-968dimer⁺CD4⁺ T cells (FIGS. 25A-25R). These T cell clones showed IL-2 production in an antigen-specific manner (FIGS. 24W and 24S). Multiple TCRαβ pairs, including one dominant pair, were isolated from both DP4^L112W/V141MRSV-GP and TT dimer⁺single-cell clones (Table 9). In FIGS. 24A-24W and 25A-25S, single-cell clones were established by limiting dilution from RSV-GP_162-175and TT_948-968dimer⁺cells. When these RSV-GP and TT dimer⁺single-cell clones were individually stained with three different DP4 multimers (DP4^L112W/V141Mdimers, wild-type DP4 tetramers, or wild-type DP4 dextramers), the DP4^L112W/V141Mdimers showed better staining of RSV-GP- (c12 and c39) and TT-specific clones (c2 and c9) than the conventional wild-type DP4 RSV-GP dextramers and wild-type DP4 TT tetramers and dextramers (FIGS. 26A-26NN).

Wild-type DQ5 and DQ5^L114W/V143Mdimers (Table 10) and DQ5^{L114W/V143M+4reps}dimers were produced and their staining of TCR-transduced CD4⁺ T cells was compared. The wild-type DQ5 dimers could not detect E6-transduced CD4⁺ T cells. The DQ5^L114W/V143Mdimers showed only weak staining of the E6-transduced CD4⁺ T cells compared to the DQ5^{L114W/V143M+4reps}dimers, which instead showed robust staining (FIGS. 27A-27L). To validate DQ5^{L114W/V143M+4reps}dimer staining, we cloned a DQ5-restricted TCR gene specific to GPC3_138-157from dimer⁺CD4⁺ T cells in vitro expanded in a peptide-specific manner. When clonotypically reconstituted in human CD4⁺ TCR-deficient T cells, the TCR was successfully stained by the cognate DQ5^{L114W/V143M+4reps}dimer and were functional in a DQ5-restricted and antigen-specific manner (FIGS. 28A-28G).

TABLE 10

TCR Sequences

No.
Peptide
TRAV
TRAJ
TCRα CDR3
TRBV
TRBJ
TCRβ CDR3

06
GPC3_138-157
9-2*02
27*01
CALYTNAGKSTF
15*02
2-3*01
CATSRDVSSTDTQYF

(SEQ ID NO: 316)

(SEQ ID NO: 317)

Ex vivo staining was performed of memory CD4⁺ T cells with DR1^{L114W/V143M+2reps}dimers specific to influenza virus hemagglutinin (Flu-HA) peptides without in vitro stimulation. A small subset of the CD4⁺ T cells were positively stained with DR1^{L114W/V143M+2reps}dimers for Flu-HA_117-136- and Flu-HA_306-318(FIGS. 29A-29L). Wild-type DR1, DR1^L114W/V143Mand DR1^{L114W/V143M+6reps}dimers and DR1^{L114W/V143M+2reps}dimers were produced and their staining of TCR-transduced CD4⁺ T cells was compared. Both wild-type DR1 and DR1^L114W/V143Mdimers detected very little of the cognate TCR (HA1.7) on CD4⁺ T cells, while DR1^{L114W/V143M+2reps}and DR1^{L114W/V143M+6reps}dimers showed similar robust staining. Importantly, DR1^{L114W/V143M+2reps}dimers stained HA1.7-transduced CD4⁺ T cells more robustly and with better separation than the wild-type DR1 dextramer (FIGS. 30A-30X). To validate DR1^{L114W/V143M+2reps}dimer staining, DR1-restricted TCR genes specific to HSD17B12_225-244and LY6K_99-118were cloned from dimer⁺ CD4⁺ T cells in vitro expanded in a peptide-specific manner. When clonotypically reconstituted in primary CD4⁺ T cells, the two TCRs (Table 11) were successfully stained by the cognate DR1^{L114W/V143M+2reps}dimers and were functional in a DR1-restricted and antigen-specific manner (FIGS. 31A-310).

TABLE 11

TCR Sequences

No.
Peptide
TRAV
TRAJ
TCRα CDR3
TRBV
TRBJ
TCRβ CDR3

07
HSD17B12_225-244
5*01
4*01
CADLSGGYNKLIF
11*01
2-3*01
CASSPTLGTDTQYF

(SEQ ID

(SEQ ID

NO: 318)

NO: 319)

08
LY6K_99-118
38-
52*01
CAYRSFLNAG
20-
1-6*01
CAASRESKWS

2/DV8*01

GTSYGKLTF
1*01

SYNSPLHF

(SEQ ID

(SEQ ID

NO: 320)

NO: 321)

Methods

Cells

Peripheral mononuclear cells were obtained via density gradient centrifugation. K562-based artificial antigen presenting cells (aAPCs) individually expressing various HLA class II genes as a single HLA allele in conjunction with CD80 and CD83 have been reported previously (see Butler, M. O. et al., PLoS One 7, e30229 (2012)). The Jurkat 76 cell line is a T cell leukemic cell line lacking endogenous TCR, CD4, and CD8 expression (see. Heemskerk, M. H. et al., Blood 102, 3530-3540 (2003)). Jurkat 76/CD4 cells were generated by retrovirally transducing the human CD4 gene. A375 cells are a melanoma cell line. HEK293T cells and A375 cells were grown in DMEM supplemented with 10% FBS and 50 μg/ml gentamicin. The Jurkat 76 cell line was cultured in RPMI 1640 supplemented with 10% FBS and 50 μg/ml gentamicin.

Peptides/Antibodies

Synthetic peptides were dissolved at 50 mg/ml in DMSO. The following antibodies were used for flow cytometry analysis: APC-Cy7-conjugated anti-CD4 (RPA-T4, BIOLEGEND, San Diego, Calif.; see Wooldridge, L. et al., Eur J Immunol 36, 1847-1855 (2006)) and PE-conjugated anti-His tag (AD1.1.10, ABCAM, Cambridge, Mass.). Dead cells were distinguished with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit. Stained cells were analyzed with FACSCanto II or LSRFortessa X-20. Cell sorting was conducted using a FACSAria II. Data analysis was performed using FlowJo software (version 9.9.6).

Genes

Novel TCR genes were cloned via 5′-rapid amplification of cDNA ends (RACE) PCR and sequenced as previously described (see, e.g., Nakatsugawa, M. et al., Sci Rep 6, 23821 (2016); Nakatsugawa, M. et al., J Immunol 194, 3487-3500 (2015); Ochi, T. et al., Cancer Immunol Res 3, 1070-1081 (2015); each of which is incorporated by reference herein in its entirety). All genes were cloned into the pMX retroviral vector and transduced into cell lines using the 293GPG and PG13 cell-based retrovirus system (see, e.g., Hirano, N. et al., Blood 107, 1528-1536 (2006); Butler, M. O. et al., Clin Cancer Res 13, 1857-1867 (2007); Hirano, N. et al., Clin Cancer Res 12, 2967-2975 (2006); each of which is incorporated by reference herein in its entirety).

Generation of the HLA Class II Monomer and Dimer

HEK293T cells were transfected with the α and β genes using the 293GPG cell-based retrovirus system (see Hirano, N. et al., Blood 107, 1528-1536 (2006); Butler, M. O. et al., Clin Cancer Res 13, 1857-1867 (2007); Hirano, N. et al., Blood 108, 2662-2668 (2006)) and cultured in DMEM supplemented with 10% FBS and 50 μg/ml gentamicin. For DP4 dimer staining, HEK293T cells stably secreting soluble DP4^L112W/V141Mprotein were grown until confluent, and the medium was changed to serum-free 293 SFM II medium (Thermo Fisher Scientific, Waltham, Mass.).

A375 cells were transfected with the α and β genes using the 293GPG cell-based retrovirus system (see, e.g., Hirano, N. et al., Blood 107, 1528-1536 (2006); Butler, M. O. et al. Clin Cancer Res 13, 1857-1867 (2007); and Hirano, N. et al., Blood 108, 2662-2668 (2006); each of which is incorporated by reference herein in its entirety) and cultured in DMEM supplemented with 10% FBS and 50 μg/ml gentamicin.

After forty-eight hours, the conditioned medium was harvested and concentrated using Amicon Ultra filters (molecular weight cut-off (MWCO) 10 kDa) (MilliporeSigma, Burlington, Mass.). The soluble HLA class II-containing supernatant was then mixed with 100 μg/ml peptide of interest for 20-24 hours at 37° C. for in vitro peptide exchange. The concentration of the monomer was measured by specific ELISA using a nickel-coated plate and an anti-His tag biotinylated mAb. Soluble HLA class II monomer was dimerized using a PE-conjugated anti-His mAb at a 2:1 molar ratio for 1.5 hours at 4° C. for staining.

Stimulation of DP4-Restricted Antigen-Specific CD4⁺T Cells

CD4⁺ T cells were purified and stimulated with DP4-expressing aAPCs pulsed with DP4-restricted peptides at 10 μg/ml and irradiated at 200 Gy at an E:T ratio of 20:1. After forty-eight hours, 10 IU/ml IL-2 and 10 ng/ml IL-15 were added to the CD4⁺ T cells. The culture medium supplemented with IL-2 (10 IU/ml) and IL-15 (10 ng/ml) was replenished every 2-3 days. After 2 weeks of stimulation, the T cells were subjected to DP4^L112W/V141Mdimer staining.

Stimulation of DQ5-Restricted Antigen-Specific CD4⁺T Cells

CD4⁺ T cells were purified and then stimulated with DQ5-expressing aAPCs pulsed with GPC3_138-157at 10 μg/ml and irradiated at 200 Gy at an E:T ratio of 20:1. After forty-eight hours, 10 IU/ml IL-2 and 10 ng/ml IL-15 were added to the CD4⁺ T cells. The culture medium supplemented with IL-2 (10 IU/ml) and IL-15 (10 ng/ml) was replenished every 2-3 days. Two weeks later, the T cells were subjected to DQ5^{L114W/V143M+4reps}dimer staining.

Stimulation of DR1-Restricted Antigen-Specific CD4⁺ T Cells

CD4⁺ T cells were purified and then stimulated with DR1-expressing aAPCs pulsed with DR1-restricted peptides at 10 μg/ml and irradiated at 200 Gy at an E:T ratio of 20:1. After forty-eight hours, 10 IU/ml IL-2 and 10 ng/ml IL-15 were added to the CD4⁺ T cells. The culture medium supplemented with IL-2 (10 IU/ml) and IL-15 (10 ng/ml) was replenished every 2-3 days. Two weeks later, the T cells were subjected to DR1^{L114W/V143M+2reps}dimer staining.

HLA Class H Dimer, Tetramer, and Dextramer Staining

DP4 tetramers and dextramers, and DR1 dextramers were compared in multimer staining analysis.

Primary CD4⁺ T cells and Jurkat 76/CD4 T cells transduced with antigen-specific TCR genes were pretreated with 50 nM dasatinib for 30 min at 37° C. and stained with 5-15 μg/ml class II dimers for 4-5 hours at room temperature. After washing, cell surface molecules were counterstained with an APC-Cy7-conjugated anti-CD4 mAb.

Dimer Staining of Unstimulated CD4⁺ T Cells from PBMCs from Melanoma Patients

One million CD4⁺ T cells were purified and pretreated with 50 nM dasatinib for 30 min at 37° C. The cells were stained with 5-15 μg/ml class II dimers for 4-5 hours at room temperature. After washing, cell surface molecules were counterstained with an APC-Cy7-conjugated anti-CD4 mAb. The absolute counts of the dimer⁺ cells were determined by flow cytometry.

Expansion of DP4 Dimer⁺ T Cells and Establishment of Single T Cell Clones.

To expand DP4^L112W/V141Mdimer⁺ T cells, CD4⁺ T cells were stimulated and stained with DP4^L112W/V141Mdimers as described above. The dimer⁺ cells were sorted by using anti-PE magnetic beads and expanded by using artificial APC/mOKT3 irradiated at 200 Gy at an E:T ratio of 5-20:1 (see Butler, M. O. et al., PLoS One 7, e30229 (2012)). The culture medium supplemented with IL-2 (10 IU/ml) and IL-15 (10 ng/ml) was replenished every 2-3 days. Two to three weeks later, the T cells were subjected to DP4^L112W/V141Mdimer staining. DP4^L112W/V141Mdimer⁺ single-cell clones were generated by limiting dilution as previously described (see Su, L. F. et al., Immunity 38, 373-383 (2013)). Briefly, memory CD4⁺ T cells were purified and stained with DP4^L112W/V141Mdimers without dasatinib pretreatment. The dimer⁺ cells were sorted and then stimulated with 5 μg/ml PHA-P and PBMCs from multiple allogeneic donors irradiated at 20 Gy in a 96-well plate. The culture medium was supplemented and replenished after 1 week of stimulation with IL-2 (100 IU/ml) and IL-15 (10 ng/ml). Two weeks later, single-cell clones were stained with DP4^L112W/V141Mdimers.

ELISPOT Assay

Cytokine ELISPOT assays were performed as previously reported (see, e.g., Yamashita, Y. et al., Nat Commun 8, 15244 (2017); and Anczurowski, M. et al., Sci Rep 8, 4804 (2018)); each of which is incorporated by reference herein in its entirety).

	Number	Date	Country
	63029103	May 2020	US
	62880492	Jul 2019	US

METHODS OF IDENTIFYING T CELL RECEPTORS

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