Methods for identifying t cell receptors

Abstract

The present disclosure relates to methods of identifying MHC class II specific T cell receptors (TCRs). In certain aspects, the methods comprise contacting a T cell with a complex comprising (i) an MHC class II molecule having a higher affinity for CD4 than a naturally occurring MHC class II molecule, and (ii) a peptide, e.g., an epitope. In certain aspects, the HLA class II molecule comprises a beta chain having one or more mutations relative to a wild-type beta chain sequence.

Description

[0001] Cross-references to related applications

[0002] This PCT application claims priority to U.S. Provisional Application No. 62 / 880,492, filed July 30, 2019, and U.S. Provisional Application No. 63 / 029,103, filed May 22, 2020, each of which is incorporated herein by reference in its entirety.

[0003] References to sequence lists submitted electronically via EFS-WEB

[0004] The contents of the sequence list submitted electronically (name: 4285.009PC02_SL_ST25.txt, size: 291,794 bytes; creation date: July 28, 2020) are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure provides a method for identifying MHC class II specific T cell receptors (“TCRs”). Background Technology

[0006] Immunotherapy has become a key tool in the fight against a variety of diseases, including cancer. T-cell therapy is at the forefront of immunotherapy development, and adoptive transfer of anti-tumor T cells has been shown to induce clinical responses in cancer patients. Although many T-cell therapies target mutated tumor antigens, the vast majority of these neoantigens are not shared and are unique to each patient.

[0007] The number of potential non-mutated antigens is orders of magnitude greater than that of mutated antigens. Elucidating T-cell epitopes derived from shared antigens could contribute to the robust development of effective and safe adoptive T-cell therapies, which could be readily applied to a larger population of cancer patients. However, the absolute number of non-mutated antigens and the high polymorphism of HLA genes may hinder a comprehensive analysis of the specificity of anti-tumor T-cell responses to non-mutated antigens. Summary of the Invention

[0008] Certain aspects of this disclosure relate to a method for identifying MHC class II specific T cell receptors (TCRs), the method 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 α chain and a β chain, wherein the MHC class II molecule has a higher affinity for CD4 than naturally occurring MHC class II molecules; and wherein the MHC class II specific TCR specifically binds to the complex comprising the MHC class II molecule and the peptide.

[0009] In some aspects, the β chain of an MHC class II molecule contains an amino acid sequence with one or more mutations relative to the wild-type β chain of the MHC class II molecule. In some aspects, the α chain of an MHC class II molecule contains an amino acid sequence with one or more mutations relative to the wild-type α chain of the MHC class II molecule. In some aspects, one or more mutations include substitution mutations.

[0010] In some respects, MHC class II molecules are HLA-DP, HLA-DQ, or HLA-DR alleles, or any combination thereof. In some respects, (i) the β chain of an HLA class II molecule is an HLA-DP allele, (ii) the α chain of an HLA class II molecule is an HLA-DP allele, or (iii) both (i) and (ii). In some respects, the β chain of an HLA class II molecule is a DP1, DP2, DP3, DP4, DP5, DP6, DP8, or DP9 allele.

[0011] In some respects, the β chain of MHC class II molecules contains HLA alleles selected from the following groups: 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*108, DPB1*109, DPB1*110, DPB1*111, DPB1*112, DPB1*113, DPB1*114, DPB1*115, DPB1*116, DPB1*117, DPB1*118, DPB1*119 ... B1*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*14 1. 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, DPB D PB1*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, D Alleles of PB1*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.

[0012] In some respects, the α chain of MHC class II molecules contains HLA-DPA1*01, HLA-DPA1*02, HLA-DPA1*03, or HLA-DPA1*04 alleles.

[0013] In some aspects, the β chain of an MHC class II molecule contains an amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO:1. In some aspects, the β chain of an MHC class II molecule contains an amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO:1.

[0014] Certain aspects of this disclosure relate to a method for identifying an MHC class II specific T cell receptor (TCR), the method 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 α chain and a β chain, wherein the β chain of the MHC class II molecule comprises (i) an amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1, (ii) an amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1, or (iii) both of (i) and (ii); and wherein the MHC class II specific TCR specifically binds to the complex comprising the MHC class II molecule and the peptide.

[0015] In some respects, MHC class II molecules have a higher affinity for CD4 than naturally occurring MHC class II molecules.

[0016] In some aspects, the amino acid at position 112 of amino acid residue SEQ ID NO:1, excluding leucine, comprises a hydrophobic side chain. In some aspects, the amino acid at position 112 of amino acid residue SEQ ID NO:1, excluding leucine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid at position 112 of amino acid residue SEQ ID NO:1, excluding leucine, is tryptophan.

[0017] In some aspects, the amino acid at position 141 of amino acid residue SEQ ID NO:1, excluding valine, comprises a hydrophobic side chain. In some aspects, the amino acid at position 141 of amino acid residue SEQ ID NO:1, excluding valine, is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid at position 141 of amino acid residue SEQ ID NO:1, excluding valine, is methionine.

[0018] In some respects, (i) the β chain of an HLA class II molecule is an HLA-DQ allele, (ii) the α chain of an HLA class II molecule is an HLA-DQ allele, or (iii) both (i) and (ii). In some respects, the β chain of an HLA class II molecule contains the DQ2, DQ3, DQ4, DQ5, or DQ6 alleles. In some respects, the β chain of an MHC class II molecule contains the HLA-DQB1*02, HLA-DQB1*03, HLA-DQB1*04, HLA-DQB1*05, or HLA-DQB1*06 alleles. In some respects, the α chain of MHC class II molecules contains alleles of HLA-DQA1*01, HLA-DQA1*02, HLA-DQA1*03, HLA-DQA1*04, HLA-DQA1*05, or HLA-DQA1*06.

[0019] In some respects, the β chain of an MHC class II molecule comprises (a) an amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11; (b) an amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11; and (c) at least three of the following: (i) an amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11; (ii) an amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11; (iii) an amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11; and (iv) an amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11.

[0020] In some respects, the β chain of an MHC class II molecule contains (a) an amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11; (b) an amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11; (c) an amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11; (d) an amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11; (e) an amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11; and (f) an amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11.

[0021] In some aspects, the amino acid at position 114 of amino acid residue SEQ ID NO:11, excluding leucine, comprises a hydrophobic side chain. In some aspects, the amino acid at position 114 of amino acid residue SEQ ID NO:11, excluding leucine, is selected from the group consisting of: alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid at position 114 of amino acid residue SEQ ID NO:11, excluding leucine, is tryptophan.

[0022] In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11 comprises a hydrophobic side chain. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11 is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11 is methionine.

[0023] In some aspects, the β chain of an MHC class II molecule contains an amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11. In some aspects, the amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11 is selected from serine, threonine, and glutamine. In some aspects, the amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11 is glutamine.

[0024] In some aspects, the β-chain of an MHC class II molecule contains an amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11. In some aspects, the amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11 is selected from alanine, valine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11 is valine.

[0025] In some aspects, the β chain of an MHC class II molecule contains an amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11 is selected from arginine, histidine, and lysine. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11 is histidine.

[0026] In some aspects, the β-chain of an MHC class II molecule contains an amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11. In some aspects, the amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11 is selected from serine, threonine, asparagine, and glutamine. In some aspects, the amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11 is glutamine.

[0027] In some respects, (i) the β chain of HLA class II molecules is an HLA-DR allele, (ii) the α chain of HLA class II molecules is an HLA-DR allele, or (iii) both (i) and (ii) are present.

[0028] In some respects, the β chain of HLA class II molecules contains alleles of DR2, DR3, DR4, DR5, DR6, DR7, DR8, DR9, DR10, DR11, DR12, DR13, DR14, DR15, or DR16. In some respects, the β chain of MHC class II molecules contains HLA alleles selected from the following groups: 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 respects, the α chain of MHC class II molecules contains the HLA-DRA1*01 allele.

[0029] In some aspects, the β chain comprises: (a) an amino acid other than leucine at position 114 corresponding to amino acid residue SEQ ID NO:19; (b) an amino acid other than valine at position 143 corresponding to amino acid residue SEQ ID NO:19; and (c) at least two of the following: (i) an amino acid other than serine at position 118 corresponding to amino acid residue SEQ ID NO:19; (ii) an amino acid other than lysine at position 139 corresponding to amino acid residue SEQ ID NO:19; (iii) an amino acid other than glycine at position 146 corresponding to amino acid residue SEQ ID NO:19; (iv) an amino acid other than threonine at position 157 corresponding to amino acid residue SEQ ID NO:19; (v) an amino acid other than threonine at position 163 corresponding to amino acid residue SEQ ID NO:19; and (vi) an amino acid other than valine at position 164 corresponding to amino acid residue SEQ ID NO:19.

[0030] In some aspects, the β chain comprises: (c) at least three of the following: (i) an amino acid other than leucine at position 118 corresponding to amino acid residue SEQ ID NO:19; (ii) an amino acid other than lysine at position 139 corresponding to amino acid residue SEQ ID NO:19; (iii) an amino acid other than glycine at position 146 corresponding to amino acid residue SEQ ID NO:19; (iv) an amino acid other than threonine at position 157 corresponding to amino acid residue SEQ ID NO:19; (v) an amino acid other than threonine at position 163 corresponding to amino acid residue SEQ ID NO:19; and (vi) an amino acid other than valine at position 164 corresponding to amino acid residue SEQ ID NO:19.

[0031] In some aspects, the β chain comprises: (c) at least four of the following: (i) an amino acid other than leucine at position 118 corresponding to amino acid residue SEQ ID NO:19; (ii) an amino acid other than lysine at position 139 corresponding to amino acid residue SEQ ID NO:19; (iii) an amino acid other than glycine at position 146 corresponding to amino acid residue SEQ ID NO:19; (iv) an amino acid other than threonine at position 157 corresponding to amino acid residue SEQ ID NO:19; (v) an amino acid other than threonine at position 163 corresponding to amino acid residue SEQ ID NO:19; and (vi) an amino acid other than valine at position 164 corresponding to amino acid residue SEQ ID NO:19.

[0032] In some aspects, the β chain comprises: (a) an amino acid other than leucine at position 114 corresponding to amino acid residue SEQ ID NO:19, (b) an amino acid other than valine at position 143 corresponding to amino acid residue SEQ ID NO:19, (c) an amino acid other than serine at position 118 corresponding to amino acid residue SEQ ID NO:19, and (d) an amino acid other than threonine at position 157 corresponding to amino acid residue SEQ ID NO:19.

[0033] In some respects, the β chain comprises: (a) an amino acid other than leucine at position 114 corresponding to amino acid residue SEQ ID NO:19, (b) an amino acid other than valine at position 143 corresponding to amino acid residue SEQ ID NO:19, (c) an amino acid other than serine at position 118 corresponding to amino acid residue SEQ ID NO:19, (d) an amino acid other than lysine at position 139 corresponding to amino acid residue SEQ ID NO:19, (e) an amino acid other than glycine at position 146 corresponding to amino acid residue SEQ ID NO:19, (f) an amino acid other than threonine at position 157 corresponding to amino acid residue SEQ ID NO:19, (g) an amino acid other than threonine at position 163 corresponding to amino acid residue SEQ ID NO:19, and (h) an amino acid other than valine at position 164 corresponding to amino acid residue SEQ ID NO:19.

[0034] In some aspects, the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19 comprises a hydrophobic side chain. In some aspects, the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19 is selected from the group consisting of: alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19 is tryptophan.

[0035] In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19 comprises a hydrophobic side chain. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19 is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19 is methionine.

[0036] In some aspects, the β-chain of an MHC class II molecule contains an amino acid other than serine at position 118 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:19 is selected from arginine, histidine, and lysine. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:19 is histidine.

[0037] In some aspects, the β-chain of an MHC class II molecule contains an amino acid other than lysine at position 139 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than lysine at position 139 of amino acid residue SEQ ID NO:19 is selected from serine, threonine, and glutamine. In some aspects, the amino acid other than lysine at position 139 of amino acid residue SEQ ID NO:19 is threonine.

[0038] In some aspects, the β-chain of an MHC class II molecule contains an amino acid other than glycine at position 146 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than glycine at position 146 of amino acid residue SEQ ID NO:19 is selected from serine, asparagine, threonine, and glutamine. In some aspects, the amino acid other than glycine at position 146 of amino acid residue SEQ ID NO:19 is glutamine.

[0039] In some aspects, the β-chain of an MHC class II molecule contains an amino acid other than threonine at position 157 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than threonine at position 157 of amino acid residue SEQ ID NO:19 is selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than threonine at position 157 of amino acid residue SEQ ID NO:19 is isoleucine.

[0040] In some aspects, the β-chain of an MHC class II molecule contains an amino acid other than threonine at position 163 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than threonine at position 163 of amino acid residue SEQ ID NO:19 is selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than threonine at position 163 of amino acid residue SEQ ID NO:19 is methionine.

[0041] In some aspects, the β-chain of an MHC class II molecule contains an amino acid other than valine at position 164 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than valine at position 164 of amino acid residue SEQ ID NO:19 is selected from serine, asparagine, threonine, and glutamine. In some aspects, the amino acid other than valine at position 164 of amino acid residue SEQ ID NO:19 is threonine.

[0042] In some aspects, the β chain comprises: (a) tryptophan at position 114 corresponding to amino acid residue SEQ ID NO:19, (b) methionine at position 143 corresponding to amino acid residue SEQ ID NO:19, (c) histidine at position 118 corresponding to amino acid residue SEQ ID NO:19, and (d) isoleucine at position 157 corresponding to amino acid residue SEQ ID NO:19.

[0043] In some respects, naturally occurring MHC class II molecules include: (a) leucine at the position of amino acid residue 112 corresponding to SEQ ID NO:1 or amino acid residue 114 corresponding to SEQ ID NO:11 or 19, (b) valine at the position of amino acid residue 141 corresponding to SEQ ID NO:1 or amino acid residue 143 corresponding to SEQ ID NO:11 or 19, or (c) both (a) and (b).

[0044] In some respects, naturally occurring MHC class II molecules comprise: (a) leucine at position 112 of SEQ ID NO:1 or 114 of SEQ ID NO:11 or 19; (b) valine at position 143 of SEQ ID NO:1 or SEQ ID NO:11 or 19; (c) asparagine at position 110 of SEQ ID NO:11; (d) isoleucine at position 116 of SEQ ID NO:11; (e) serine at position 118 of SEQ ID NO:11 or 19; and (f) proline at position 146 of SEQ ID NO:11; (g) lysine at position 139 of SEQ ID NO:19; (h) glycine at position 146 of SEQ ID NO:19; and (i) valine at position 114 of SEQ ID NO:11 or 19. (j) threonine at position 157 of amino acid residue NO:19, (k) threonine at position 163 of amino acid residue NO:19, (l) valine at position 164 of amino acid residue NO:19, or any combination of (a) to (k).

[0045] In some respects, MHC class II molecules are dimers. In some respects, MHC class II molecules are trimers. In some respects, MHC class II molecules are tetramers. In some respects, peptides contain fragments of proteins. In some respects, proteins are expressed by diseased cells. In some respects, proteins are expressed by tumor cells.

[0046] In some respects, a peptide contains at least about 10 amino acids. In other respects, a peptide contains 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.

[0047] In some respects, peptides contain approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids.

[0048] In some respects, MHC class II molecules are expressed on the surface of antigen-presenting cells.

[0049] In some respects, T cells are obtained from human subjects. In other respects, T cells are tumor-infiltrating lymphocytes (TILs).

[0050] In some respects, the affinity of MHC class II molecules for CD4 is 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 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, or at least about 100.

[0051] In some aspects, the method further includes selecting T cells bound by MHC class II molecules. In some aspects, the method further includes isolating TCRs bound by MHC class II molecules. In some aspects, the method further includes sequencing the TCRs. In some aspects, the method further includes cloning the TCRs. In some aspects, the method further includes recombinantly expressing the TCRs in host cells.

[0052] In some respects, MHC class II molecules have K values ​​of less than about 100 μM, less than about 50 μM, less than about 20 μM, or less than about 10 μM. D Binding to CD4. In some respects, MHC class II molecules bind with K at approximately 14 μM or less. D Binding to CD4. In some respects, MHC class II molecules bind at approximately 8.9 μM or less K. D Combined with CD4. Attached Figure Description

[0053] Figures 1A to 1V illustrate the DP4 affinity maturation process. L112W / V141M Graphical representation of data showing enhanced CD4 binding ability of the molecules. Figures 1A to 1F are histograms showing the results of DP4 substitution with blank, wild-type, or DP4-containing L112W, V114M, V141M, and M158I. L112W / V114M / V141M / M158I Results of HLA class II-empty K562 cells stably expressing wild-type DPα chain (DPA1*01:03) transduced with mutant DPβ chain (DPB1*04:01) and stained with anti-class II mAb and soluble CD4 (sCD4). Figure 1G This is a bar chart summarizing the binding affinity (MFI; y-axis) of sCD4 to all possible DP4 reversion mutants, which are expressed similarly to those in Figures 1A through 1F and stained with sCD4. As quantified by steady-state analysis, Figure 1H DP4 was displayed L112W / V141M Affinity with CD4. Figure 1IThis shows the expression of wild-type DP4 or DP4 by pulses with graded concentrations of DP4 / WT1 peptide. L112W / V141M The results of IL-2 EPISPOT assays in DP4 / WT1 TCR clone 9 transduced Jurkat76 and Jurkat 76 / CD4 cells stimulated by aAPC. Figures 1J to 1W show the expression of DP4 / WT1 TCR clone 9 cells stained with anti-class II mAb and sCD4. L112W / V141M Histograms of K562 cells with alleles (as shown). Open histograms represent isotype control staining. *P < 0.05, obtained by Student's t-test. Bars and error bars represent the mean ± SD of triplicate experimental results. At least two independent experiments were performed. Figures 1X through 1AA are histograms showing wild-type DP4 and DP4L112W / V141M molecules detected on the surface of K562 cells with indicated anti-HLA class II antibodies. Control cells without class II expression are shown as solid gray. Figures 1AB through 1BH are histograms showing aAPCs expressing indicated DP4 or class II parental cells stained with sCD4, indicating concentrations. Figure 1 BI The expression concentration of wild-type DP4 or DP44 is shown. L112W / V141M The quantification of aAPC. Error bars represent the mean ± standard deviation of triplicate experiments. Figure 1BJ This is a biolayer interferometric sensing image showing the interaction between biotinylated wild-type DP4 (ligand) and sCD4 (analyte) within a certain concentration range. Figure 1BK This is a biolayer interferometric sensing image showing biotinylated DP4 within a certain concentration range. L112W / V141M Interaction between (ligand) and sCD4 (analyte). Figure 1BJ and 1BI The experiments were conducted in parallel. All data represent two independent experiments.

[0054] Figures 2A to 2D It is DP4 L112W / V141M A banded diagram of the model structure of the human CD4 complex. Figures 2A to 2B These are two orientations of the ternary complex model structure of DPA1*01:03, DPB1*04:01, and CD4, as shown in the diagram. The DPB1*04:01-CD4 binding interface is enclosed in a dashed box. Figure 2B ). Figures 2C to 2D Wild-type DP4 is provided Figure 2C ) and DP4 L112W / V141M ( Figure 2D A close-up view of the CD4 binding interface. The side chains of the interacting residues are shown in ball-and-stick representation. Figures 2C to 2D ).

[0055] Figures 3A to 3P illustrate DP4. L112W / V141MDimer staining in human primary CD4 + Graphical representation of data on homologous TCR expression in T cells. Using DP4 / MAGE-A3. 243-258 (R12C9; Figures 3E to 3H), DP4 / WT1 328-348 (Clone 9; Figures 3I to 3L) or DP4 / NY-ESO-1 157-170 (5B8; Figs. 3M to 3P) TCR transduction of primary T cells with indicated DP4 L112W / V141M Dimer staining (Figs. 3B to 3D, 3F to 3H, 3J to 3L and 3N to 3P).

[0056] Figures 4A to 4D illustrate the use of DP4 L112W / V141M Dimer and R12C9 transduced CD4 transduction with anti-Vβ22 mAb staining + Scatter plot of complex staining of T cells. Note that R12C9 represents Vβ22. Figures 4E to 4H illustrate the use of DP4. L112W / V141M CD4 clone 9 transduced with dimer and anti-NGFR mAb double staining + Scatter plot of complex staining of T cells. Note the fusion of clone 9 and the ΔNGFR gene with P2A.

[0057] Figures 5A to 5P illustrate the use of 5 μg / ml conventional wild-type DP4 tetramer and DP4 L112W / V141M Scatter plots of primary T cells stained with a combination of clone 9 transduction (Fig. 5A to 5H) and 5B8 transduction (Fig. 5I to 5P) for dimer staining. Perform at least two independent experiments.

[0058] Figures 6A to 6F This explains the use of DP4 L112W / V141M A bar graph showing the results of comprehensive screening of dimers, which identified a range of novel DP4-restricted tumor-associated antigens. From 6 DP4... + Purified peripheral CD4 from melanoma patients + T cells were stimulated with aAPCs expressing DP4 by shocks with 196 different peptides derived from tumor-associated antigens, and homologous DP4 was used. L112W / V141M Dimer staining. Results were displayed using the 30 peptides with the highest positive values. Figures 6A to 6B The results for the remaining 166 peptides were shown in... Figures 6C to 6F In the middle. Set each gate so that the control dimer staining shows <0.2% positivity. Positive dimer staining is defined as staining exceeding 3 standard deviations of control dimer staining, as shown by the dashed line (>0.6%).

[0059] Figures 7A to 7L show peptide-specific CD4 from melanoma patients. + DP4 of T cells L112W / V141M Graphical representation of dimer staining. From 6 DP4 + Purified primary CD4 from melanoma patients + T cells were stimulated with aAPCs expressing DP4 by shocks with 196 different peptides derived from tumor-associated antigens, and homologous DP4 was used. L112W / V141M Dimer staining, such as Figures 6A to 6F As shown. DP4 is displayed. L112W / V141M Example of dimer staining. *P<0.05, obtained by Student's t-test. ns, not significant. Perform at least two independent experiments.

[0060] Figures 8A to 8X are graphical representations of the data, illustrating the changes from DP4. L112W / V141M Isolate from dimeric positive cells and in human TCR-deficient CD4 + The reconstructed DP4-restricted TCR in T cells functions in a DP4-restricted and antigen-specific manner. 03-CCND1 219-238 (Figures 8A to 8D), 05-HSD17B12 225-244 and 09-HSD17B12 225-244 (Figures 8E to 8J), 05-LGSN 296-315 (Figures 8K to 8N), 03-MAGE-A2 108-127 and 06-MAGE-A2 108-127 (Figures 8O to 8T) and 05-MUC5AC 4922-4941 (Figures 8U to 8X) From DP4 L112W / V141M Dimer-positive cell clones were reconstituted in TCR-deficient Jurkat76 / CD4 cells and used with the corresponding DP4. L112W / V141M Dimer staining.

[0061] Figures 9A to 9G This is a bar graph illustrating the O3-CCND1 levels stimulated by αAPC with the corresponding peptide in the IL-2 ELISPOT assay. 219-238 ( Figure 9A ), 05-HSD17B12 225-244 ( Figure 9B ), 09-HSD17B12 225-244 ( Figure 9C ), 05-LGSN 296-315 ( Figure 9D ), 03-MAGE-A2 108-127 ( Figure 9E ), 06-MAGE-A2 108-127 ( Figure 9F ) and 05-MUC5AC4922-4941 ( Figure 9G Results of IL-2 EPISPOT assays were obtained. DP4 / WT1 (clone 9) TCR was used as a negative control. At least two independent experiments were performed. *, P < 0.05, obtained by Student's t-test. Bars and error bars represent the mean ± SD of three experimental results.

[0062] Figure 10A to Figure 10Q It is a graphical representation of the data, showing the data from DP4. L112W / V141M Isolate from dimer-positive cells and in human primary CD4 cells + The reconstructed DP4-restricted TCR in T cells functions in a DP4-restricted and antigen-specific manner. 03-CCND1 219-238 (Figures 10A to 10D and) Figure 10O ), 03-MAGE-A2 108-127 and 06-MAGE-A2 108-127 (Figures 10E to 10J and) Figure 10P ) and 05-MUC5AC 4922-4941 (Figures 10K to 10N and) Figure 10Q Transduced into human primary CD4 by a retrovirus + T cells and with corresponding DP4 L112W / V141M Dimer staining (Figs. 10A to 10N). *P<0.05, obtained by Student's t-test. ns, not significant. Perform at least two independent experiments. *,P<0.05, obtained by Student's t-test. Bars and error bars represent the mean ± SD of three experimental results.

[0063] Figures 11A to 11E The presented data show that DP4-restricted TCRs from melanoma patient clones recognize peptides that are endogenously processed and presented by K562-based aAPCs. Figures 11A to 11B This demonstrates the endogenous expression of CCDN1 in K562-derived aAPC cells. Figure 11A ) and MAGE-A2 ( Figure 11B Images of gel chromatography. Figures 11C to 11D It shows 03-CCND1 219-238 ( Figure 11C ) or 06-MAGE-A2 108-127 ( Figure 11D Bar graph showing the results of IFN-γ ELISPOT assays in human primary T cells transduced with retroviral agents and stimulated with HLA-empty or DP4-aAPC cells without peptide shock. Figures 11C to 11D ). Figure 11E It shows that it uses 05-MUC5AC 4922-4941 TCR retroviral transduction and MUC5AC 4914-4949Bar graphs of IFN-γ ELISPOT assays in small gene transduction and HLA-empty or DP4-aAPC-unstimulated human primary T cells without peptide shock. At least two independent experiments were performed. *, P < 0.05, obtained via Student's t-test. Bars and error bars represent the mean ± SD of three experimental results.

[0064] Figure 12A The data presented in Figure 12E is 06-MAGE-A2 108-127 TCR recognizes melanoma cell lines in a DP4 and MAGE-A2 dependent manner. Figure 12A These are Western blot images showing endogenous MAGE-A2 expression in K562 cells and the melanoma cell line shown. Figures 12B to 12E are bar charts showing the expression of MAGE-A2 in K562 cells and the melanoma cell line shown. + MAGE-A2 - (Figure 12B) or SK-MEL-37 (DP4) + MAGE-A2 + (Figure 12C) Stimulation with 06-MAGE-A2 108-127 TCR-transduced primary human T cells and those using SK-MEL-28 (DP4) - MAGE-A2 + ; Figure 12D) and Me275 (DP4) - MAGE-A2 + (Figure 12E) Data from IFN-γ ELISPOT assays with DP4 transduction. *, P < 0.05, obtained by Student's t-test. Bars and error bars represent the mean ± SD of triplicate experimental results. At least two independent experiments were performed.

[0065] Figures 13A to 13Q are histograms comparing the expression levels of wild-type HLADP*04:01 and its derivatives in K562 cells stained with anti-HLA class II mAb clone 9-49. Open histograms represent isotype control staining.

[0066] Figures 14A to 14F Data are provided illustrating the enhanced CD4 binding ability of the modified DQ molecule. Figure 14A It compares DPB1*04:01, DQB1*05:01, and DQB1*05:01. L114W / V143M+4reps A table of amino acid sequences, with mutated amino acids underlined. Figure 14B and Figure 14C Stable expression of wild-type DQ5 (DQA1*01:01 / DQB1*05:01) and DQ5 using sCD4 staining L114W / V143M DQ5 L114W / V143M+4reps Wild-type DP4 or DP4L112W / V141M (like Figure 14A A graphical representation of data for type II defective K562 cells (shown). Figure 14D This shows DQ5 cells similarly stained with sCD4, each expressing a single amino acid inversion at one of the four positions. L114W / V143M+4reps CD4 binding ability of a series of K562 derivatives of the mutant. Figure 14E This is a table listing the amino acid sequences of DPB1*04:01, DQB1*02:01, DQB1*04:02, and DQB1*06:01, with substituted 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 encodes Val at position 114. Figure 14F Graphical representations are provided showing the data on L114W / V143M+3reps substitution enhancements of DQ2, DQ4, and DQ6 binding to CD4 in the β chain. At least two independent experiments were conducted. *, P < 0.05, obtained via Student's t-test. Bars and error bars represent the mean ± SD of triplicate experimental results.

[0067] Figures 15A to 15B This is a graphical representation illustrating the detection of affinity-matured DQ dimers in homologous TCRs expressed in human primary CD4+ T cells. It shows the DQ5(DQA1*01:01-DQB1*05:01)-restricted DDX3Y-specific TCR (E6) (…). Figure 15A ) and DQ6 (DQA1*01:02-DQB1*06:02) restricted influenza virus HA-specific TCR (DM2) ( Figure 15B Reconstructed in primary human CD4+ T cells, and administered via DQ5. L114W / V143M+4reps and DQ6 L114W / V143M+3reps Dimer staining. Perform at least two independent experiments.

[0068] Figures 16A to 16Q are histograms illustrating the equivalent expression levels of HLA class II genes. HLA-DQ and its derivatives were reconstructed in K562 cells and stained with anti-HLA class II monoclonal antibodies. Anti-HLA class II monoclonal antibody clone 9-49(I3) (DQ5 and DQ6) or anti-class II monoclonal antibody clone... (DQ2 and DQ4) Surface expression of each DQ2, DQ5, and DQ6 allele was detected. Open histograms represent isotype control staining.

[0069] Figures 17A to 17F Data are provided illustrating the enhanced CD4 binding ability of the modified DR molecule. Figure 17AIt compares DPB1*04:01, DRB1*01:01, and DRB1*01:01. L114W / V143M+6reps A table of amino acid sequences, with mutated amino acids underlined. Figure 17B and Figure 17C Using wild-type DR1 (DRA1*01:01 / DRB1*01:01), DR1 L114W / V143M DR1 L114W / V143M+6reps Wild-type DP4 or DP4 L112W / V141M Graphical representation of data from stable transduced and sCD4-stained type II defective K562 cells. Figures 17D to 17E The image shows DR1 with a single amino acid inversion at one of the six positions. L114W / V143M+6reps Mutants (similarly stained with sCD4) Figure 17D ) and DRB1 carrying S118H and T157I along with L114W / V143M L114W / V143M+2reps ( Figure 17E CD4 binding ability of a series of K562 derivatives. Figure 17F This is a table listing the amino acid sequence of DPB1*04:01, and it also lists the DRB1 alleles of DR3, DR4, DR7, DR10, DR11, and DR13 with DRB1. L114W / V143M+6reps and DRB1 L114W / V143M+2reps Alleles were compared together, with mutated amino acids underlined. Figures 17G to 17L are graphical representations showing that the L114W / V143M+2reps mutation enhances the binding of DR3, DR4, DR7, DR10, DR11, and DR13 to CD4 more effectively than the L114W / V143M+6reps mutation. At least two independent experiments were performed. *, P < 0.05, obtained by Student's t-test. Bars and error bars represent the mean ± SD of triplicate experimental results. Figures 17M to 17N This is a biolayer interferometry (BII) image showing the interaction between biotinylated HLA-DR1 (ligand) and soluble CD4 (analyte) within a certain concentration range. Wild-type DR1 ( Figure 17M ) and DR1 L114W / V143M+2reps ( Figure 17N The binding experiments were conducted in parallel with wild-type DR1 ( Figure 17M No binding was detected. Figure 17O This shows the DR1 quantified through steady-state analysis. L114W / V143M+2reps A graph of affinity between CD4 and CD4. All data represent two independent experiments.

[0070] Figures 18A to 18DThis is a graphical representation illustrating the expression of homologous TCRs detected by affinity-matured DR dimers in human primary CD4+ T cells. DR1-restricted TCRs (HA1.7 and SB95) are shown. Figure 18A ), DR7-restricted TCR (SD334) Figure 18B ) and DR11-restricted TCR (F24) ( Figure 18C Reconstructed in primary human T cells, and through the corresponding DR L114W / V143M+2reps Dimer staining. Using DR11. L114W / V143M+2reps Dimer and anti-Vβ22 mAb staining of DR11-restricted F24-transduced CD4 + T cells ( Figure 18D Note that F24 represents Vβ22. Perform at least two independent experiments.

[0071] Figures 19A to 19D It is HLA-DR1 L114W / V143M+2reps A diagram of the model structure of the human CD4 complex. As shown, Figure 19A An overview of the banded model structure of the ternary complex of DRA1*01:01, DRB1*01:01, and CD4 is provided. Figures 19B to 19D Wild-type DR1 (left) and mutant DR1 are provided. L114W / V143M+2reps A close-up view of the following four mutated residues (right): L114W and V143M. Figure 19B S118H Figure 19C ) and T157I ( Figure 19D (e.g., using a ball stick for illustration).

[0072] Figures 20A to 20II are histograms illustrating the equivalent expression levels of HLA class II genes. HLA-DR and its derivatives were reconstructed in K562 cells and stained with an anti-HLA class II monoclonal antibody. Surface expression of all DR alleles was detected using the anti-HLA class II monoclonal antibody clone 9-49(I3). Open histograms represent isotype control staining.

[0073] Figures 21A to 21D show the comparison of DP4. L112W / V141M Dimers and dextramers affect endogenous TRPC1 578-597 Specific CD4 + Graphical representation of T cell staining data. This was achieved through peptide shock and irradiated DP4. + Artificial APC stimulation amplifies endogenous (non-transduced) TRPC1 in melanoma patients. 578-597 Specific CD4 + T cells and DP4 L112W / V141M TRPC1 578-597Dimer (Figure 21B) or TRPC1 578-597 Dextramer staining (Fig. 21D). The corresponding CLIP multimer was used as a control (Fig. 21A and Fig. 21C).

[0074] Figures 22A to 22F show the comparison of DP4. L112W / V141M Dimers, along with regular DP4 tetramers and dextramers, affect endogenous NY-ESO-1. 157-170 Graphical representation of specific T cell staining data. From DP4 4 + Purification of CD4 from healthy donors + T cells, and with NY-ESO-1 157-170 Impacted and irradiated DP4 + One artificial APC stimulation. For example, using three different DP4 multimers (DP4... L112W / V141M The dimer (Fig. 22B), DP4 tetramer (Fig. 22D), or DP4 dextramer (Fig. 22F) are shown, respectively, for the amplified CD4 + T cells were stained.

[0075] Figures 23A to 23Y show the process of using DP4 L112W / V141M Pathogen-specific CD4 staining in vitro using dimers + Graphical representation of T cell data. From five DP4 + Purified memory CD4 from donor + T cells, targeting the following pathogen-associated peptides with DP4 in the absence of in vitro stimulation. L112W / V141M In vitro staining of dimers: TT 948-968 (Figures 23F to 23J), HSV-2-UL21 283-302 (Figures 23K to 23O), Flu-HA 527-546 (Figures 23P to 23T) and RSV-GP 162-175 (Figures 23U to 23Y). CLIP peptide was used as a negative control (Figures 23A to 23E).

[0076] Figure 24A to Figure 24W It is displayed from DP4 L112W / V141M Dimer + Endogenous RSV-GP successfully established in cells 162-175 Specific CD4 + Graphical representation of T cell clone data. From DP4 No. 6. + Purified memory CD4 from donor + T cells, and DP4 without external stimulation. L112W / V141M RSV-GP 162-175The dimer was stained in vitro. Then, the dimer was cloned by limiting dilution. + CD4 + T cells. Figures 24A to 24V are graphical representations of representative dimeric staining data from 10 dimeric-positive and 1 dimeric-negative single-cell clones. 77 of the 84 clones (91.7%) were successfully stained with DP4. L112W / V141M RSV-GP 162-175 Dimer staining. Figure 24W It displays RSV-GP 162-175 Dimer + Bar graph showing antigen-specific IL-2 production in a single-cell clone.

[0077] Figure 25A to Figure 25S It is displayed from DP4 L112W / V141M Dimer + Endogenous DP4 TT successfully established in cells 948-968 Specific CD4 + Graphical representation of T cell clone data. From DP4, number 04. + Purified memory CD4 from donor + T cells, and DP4 without external stimulation. L112W / V141M TT 948-968 The dimer was stained in vitro. Then, the dimer was cloned by limiting dilution. + CD4 + T cells. Figures 25A to 25R are graphical representations of representative dimeric staining data from eight dimeric-positive and one dimeric-negative single-cell clones. 26 of the 29 clones (89.7%) were successfully stained with DP4. L112W / V141M TT 948-968 Dimer staining. Figure 25S It displays TT 948-968 Dimer + Bar graph showing antigen-specific IL-2 production in a single-cell clone.

[0078] Figures 26A to 26NN show the RSV-GP (Figures 26A to 26P) and TT (Figures 26O to 26NN) dimers. + Graphical representation of DP4 multimer staining in single-cell clones. RSV-GP dimer. + Single-cell clones (c6, c12, c26, and c39) were produced using DP4. L112W / V141M RSV-GP 162-175 Dimers (Fig. 26B, Fig. 26D, Fig. 26F, and Fig. 26H) or wild-type DP4dextramers (Fig. 26J, Fig. 26L, Fig. 26N, and Fig. 26P) were stained. TT dimers were stained. +Single-cell clones (c2, c4, c6, and c9) were prepared using three different DP4 TT solutions. 948-968 Polymer (DP4) L112W / V141M Dimer (Fig. 26R, Fig. 26T, Fig. 26V and Fig. 26X), wild-type DP4 tetramer (Fig. 26Z, Fig. 26BB, Fig. 26DD and Fig. 26FF) and wild-type DP4 dextramer (Fig. 26HH, Fig. 26JJ, Fig. 26LL and Fig. 26NN) staining.

[0079] Figures 27A to 27L show DQ5. L114W / V143M+4reps Dimer robust staining E6-transduced CD4 + Graphical representation of T cells. E6 in CD4 + Reconstruction in T cells, followed by wild-type DQ5 (Figs. 27D and 27J), DQ5 L114W / V143M (Figures 27E and 27K) and DQ5 L114W / V143M+4reps (Figures 27F and 27L) CLIP control dimer (Figures 4D to 4F) and DDX3Y 171-190 Specific dimer staining (Fig. 27J to Fig. 27L). Control cells not transduced with TCR are shown in Fig. 27A to Fig. 27C and Fig. 27G to Fig. 27I.

[0080] Figure 28A to Figure 28G This is a graphical representation showing the restriction TCR of the affinity-matured dimeric clone DQ5. From DQ5.1 + Purified primary CD4 from melanoma patients + T cells, and with irradiated GPC3 138-157 The expression of DQ5.1 was stimulated by aAPC. Two weeks later, homologous GPC3 was used. 138-157 -DQ5 L114W / V143M+4reps Dimer response to CD4 stimulation + T cells were stained (Figs. 28A-28B). GPC3-specific TCRs were reconstructed in TCR-deficient Jurkat76 / CD4 cells and stained using the corresponding DQ5 assay. L114W / V143M+4reps Dimer (Figure 28C (E6 / Control); Figure 28D (E6 / GPC3) 138-157 Figure 28E (DQ5-06-GPC3) 138-157 / Comparison); and Figure 28F (DQ5-06-GPC3) 138-157 / GPC3 138-157 )) staining. In the IL-2ELISPOT assay, Jurkat 76 / CD4 cells expressing GPC3-specific TCRs were stimulated with DQ5-K562 cells pulsed with the corresponding peptide ( Figure 28G ).

[0081] Figures 29A to 29L show the experience using DR1. L114W / V143M+2reps In vitro staining of influenza virus hemagglutinin-specific peripheral CD4-dimers + Graphical representation of T cells. From two DR1 cells. + Purified memory CD4 from donors (07 (Figs. 29A to 29F) and 08 (Figs. 29G to 29L)) + T cells, and without external stimulation, were treated with Flu-HA. 5-24 (Figures 29B and 29H), Flu-HA 117-136 (Figures 29C and 29I), Flu-HA 232-251 (Figures 29D and 29J), Flu-HA 268-287 (Figures 29E and 29K) and Flu-HA 306-318 (Figures 29F and 29L) Influenza virus hemagglutinin (Flu-HA) peptides have specific DR1. L114W / V143M+2reps Dimer staining. CLIP peptide was used as a negative control (Fig. 29A and Fig. 29G).

[0082] Figures 30A to 30X show DR1. L114W / V143M+6reps and DR1 L114W / V143M+2reps Dimer robust staining of HA1.7 transduced CD4 + Graphical representation of T cells. HA1.7 in primary CD4 cells. + Reconstruction in T cells, followed by transduction of the TCR without transduction (Figs. 30I to 30L) and with HA1.7 TCR transduction (Figs. 30M to 30P), using wild-type DR1 (Figs. 30I and 30M), DR1 L114W / V143M (Figures 30J and 30N), DR1 L114W / V143M+6reps (Figures 30K and 30O) and DR1 L114W / V143M+2reps (Figures 30L and 30P) Dimer staining, using CLIP dimer as a negative control (Figures 30A to 30H). Additionally, HA1.7 was stained in primary CD4 cells. + Reconstruction in T cells, followed by Flu-HA 306-318 Specific DR1 L114W / V143M+2reps Dimer (Fig. 30O to Fig. 30T) or wild-type DR1 dextramer (Fig. 30U to Fig. 30X) staining.

[0083] Figure 31A to Figure 31O This is a graphical representation showing the data from the affinity-matured dimeric clone DR1-restricted TCR. From two DR1... + Purified primary CD4 from melanoma patients + T cells, and with irradiated HSD17B12 225-244Impact (Figure 31B) and LY6K 99-118 Impact (Figure 31D) on aAPC stimulation of DR1 expression. Two weeks later, homologous DR1... L114W / V143M+2reps Dimer response to CD4 stimulation + T cells were stained (Figs. 31A to 31D). DR1-restricted TCRs were stained in primary CD4 cells. + Remodeling in T cells, as demonstrated by corresponding dimer staining (Figs. 31E to 31M). In the IL-2 ELISPOT assay, DR1-restricted DR1-07-HSD17B12 was used. 225-244 ( Figure 31N ) and DR1-08-LY6K 99-118 ( Figure 31O TCR's original CD4 + T cells were generated using HSD17B12. 225-244 ( Figure 31N ) and LY6K 99-118 ( Figure 31O DR1-K562 cells stimulated by peptide shock. Detailed Implementation

[0084] This disclosure relates to a method for identifying MHC class II specific TCRs, the method 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 naturally occurring MHC class II molecules. In some aspects, the MHC class II molecule comprises an α chain and a β chain, wherein the β chain of the MHC class II molecule comprises one or more mutated amino acid sequences relative to the wild-type β chain of the MHC class II molecule.

[0085] I. Terminology

[0086] To make this disclosure more readily understandable, certain terms are first defined. As used in this application, each of the following terms shall have the meaning described below unless expressly specified herein. Further definitions are set forth throughout the application.

[0087] It should be noted that the term "a / an" refers to one or more of the entities described; for example, "a nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a," "one or more," and "at least one" are used interchangeably herein.

[0088] Furthermore, when used herein, “and / or” should be considered as each of the two specified features or components disclosed herein, with or without the other. Thus, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: 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).

[0089] The term "about" is used in this document to mean approximately, roughly, or around. When used with a numerical range, it modifies the range by extending the boundaries above and below the value. Generally, the term "about" is used in this document to modify a value by a variation of about 10% above or below the value (increase or decrease).

[0090] It should be understood that whenever an aspect is described in this document as “comprising”, other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided.

[0091] Unless otherwise defined, 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 relates. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many terms in this disclosure.

[0092] Units, prefixes, and symbols are represented in their accepted International System of Units (SI) forms. Numerical ranges include the values ​​defining the range. Unless otherwise indicated, nucleotide sequences are written from left to right with a 5' to 3' orientation. Amino acid sequences are written from left to right with an amino to carboxyl orientation. The headings provided herein are not intended to limit the various aspects of this disclosure, which are derived from the entire specification. Therefore, the terms defined below are more fully defined by reference to the entire specification.

[0093] "Administration" means the physical introduction of an agent into 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 administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a method of administration other than enteral and local administration, typically by injection, and including, but not limited to, intravenous, intramuscular, intraarterial, intrasheath, intralymphatic, intralesional, intracapsular, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as intracorporeal electroporation. In some aspects, the formulation is administered via non-parenteral routes, such as oral administration. Other non-parenteral routes include local, epidermal, or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or local administration. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0094] As used herein, the term “T-cell receptor” (TCR) 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 their antigen-binding portions; chimeric TCRs; TCR fusion constructs; and synthetic TCRs. In humans, TCRs are expressed on the surface of T cells, and they are responsible for T-cell recognition and targeting by antigen-presenting cells. Antigen-presenting cells (APCs) display foreign protein (antigen) fragments that are complexed with the major histocompatibility complex (MHC) (also referred to herein as complexed with HLA molecules, such as HLA class II molecules). TCRs recognize and bind to the peptide:HLA complex and recruit CD8 (for MHC class I molecules) or CD4 (for MHC class II molecules), thereby activating the TCR. Activated TCRs initiate downstream signaling and immune responses, including the disruption of EPCs.

[0095] Typically, a TCR may consist of two chains, an α-chain and a β-chain (or less commonly, a γ-chain and a δ-chain), interconnected by disulfide bonds. Each chain contains variable regions (α-chain variable domains and β-chain variable domains) and constant regions (α-chain constant regions and β-chain constant regions). The variable domains are located distal to the cell membrane and interact with antigens. The constant regions are located proximal to the cell membrane. A TCR may further include a transmembrane region and a short cytoplasmic tail. As used herein, the term "constant region" includes the transmembrane region and the cytoplasmic tail, as well as (where present) the conventional "constant region".

[0096] The variable domains can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which contain more conserved regions called framework regions (FRs). Each α-chain and β-chain variable domain contains three CDRs and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Each variable domain contains a binding domain that interacts with the antigen. Although all three CDRs on each chain are involved in antigen binding, CDR3 is considered the primary antigen-binding region, while CDR1 and CDR2 are considered to primarily recognize HLA molecules.

[0097] Unless explicitly stated otherwise, the term "TCR" also includes antigen-binding fragments or portions of any TCR disclosed herein, and includes monovalent and bivalent fragments or portions, as well as single-chain TCRs. The term "TCR" is not limited to naturally occurring TCRs that bind to the surface of T cells. As used herein, the term "TCR" further refers to TCRs expressed on the surface of cells other than T cells (e.g., cells naturally expressing or modified to express CD4 as described herein) or TCRs that do not have a cell membrane (e.g., isolated or soluble TCRs) as described herein.

[0098] "Antigen-binding molecule," "part of TCR," or "TCR fragment" refers to any portion of the TCR smaller than the whole. Antigen-binding molecules may include antigenic CDRs.

[0099] “Antigen” refers to any molecule, such as a peptide, that elicits an immune response or is capable of being bound by a TCR. As used herein, “epitope” refers to a portion of a polypeptide that elicits an immune response or is capable of being bound by a TCR. An immune response may involve the production of antibodies or the activation of specific immune-active cells, or both. Those skilled in the art will readily understand that any macromolecule (including virtually all proteins or peptides) can be used as an antigen. Antigens and / or epitopes can be expressed endogenously, i.e., through genomic DNA, or they can be recombinantly expressed. Antigens and / or epitopes can be specific to a particular tissue, such as diseased cells, such as cancer cells, or they can be widely expressed. Furthermore, fragments of larger molecules can act as antigens. In one respect, the antigen is a tumor antigen. Epitopes can be present in longer polypeptides (e.g., proteins), or epitopes can exist as fragments of longer polypeptides. In some respects, epitopes are complexed with the major histocompatibility complex (MHC) (also referred to herein as complexed with HLA molecules, such as HLA class 1 molecules).

[0100] The term "autologous" refers to any material derived from the same individual and subsequently reintroduced into that individual. For example, autologous T-cell therapy involves administering T cells isolated from the same individual to a subject. The term "allogeneic" refers to any material derived from one individual and subsequently introduced into another individual of the same species. For example, allogeneic T-cell transplantation involves administering T cells obtained from a donor other than the subject to a subject.

[0101] As used in this article, “CCND1,” “G1 / S-specific cyclin-D1,” “B-cell lymphoma 1 protein,” “BCL-1,” or “PRAD1” refers to the human regulatory component of the cyclin D1-CDK4 (DC) complex, which phosphorylates and inhibits members of the retinoblastoma (RB) protein family, including RB1, and regulates the cell cycle during the G1 / S transition. Phosphorylation of RB1 allows the transcription factor E2F to dissociate from the RB / E2F complex and subsequently transcribe E2F target genes responsible for G1 phase progression. CCND1 is also involved in the hypophosphorylation of RB1 in early G1 phase. The cyclin D-CDK4 complex is a major integrator of various pro-mitotic and anti-mitotic signals. CCND1 is also a substrate of 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, and is essential for the nuclear translocation and activity of the cyclin D-CDK4 complex. Furthermore, CCND1 exhibits transcriptional co-repressive activity with INSM1 in a cell cycle-dependent manner at the NEUROD1 and INS promoters. Mutations, amplifications, and overexpressions of CCND1 alter cell cycle progression, which is frequently observed in various tumors and may contribute to tumorigenesis.

[0102] As used herein, CCND1 refers not only to the full-length canonical sequence but also to its variants and fragments. The amino acid sequence of CCND1 (SEQ ID NO:27) is provided in Table 1A (UniProtKB–P24385).

[0103] Table 1A. Amino acid sequence of CCND1

[0104]

[0105] As used in this article, “MUC5AC” or “mucin 5AC” refers to a human gel-forming glycoprotein of the gastric and respiratory epithelium that protects the mucosa from infection and chemical damage by binding to inhaled microorganisms and particles that are subsequently removed by the mucociliary system.

[0106] As used herein, MUC5AC refers not only to the full-length canonical sequence but also to its variants and fragments. The amino acid sequence of MUC5AC (SEQ ID NO:28) is provided in Table 1B (UniProtKB–P98088).

[0107] Table 1B. Amino acid sequence of MUC5AC

[0108]

[0109]

[0110]

[0111] As used herein, “MAGE-A2,” “melanoma-associated antigen 2,” or “cancer / testis antigen 1.2” refers to human proteins primarily expressed by tumor cells. MAGE-A2 reduces p53 / TP53 transactivation by recruiting HDAC3 to the p53 / TP53 transcription site. MAGE-A2 inhibits p73 / TP73 activity. In vitro, MAGE-A2 promotes cell viability in melanoma cell lines. MAGE-A2 is expressed in several types of tumors, such as melanoma, head and neck squamous cell carcinoma, lung cancer, and breast cancer. However, in healthy tissues, MAGE-A2 is expressed only in the testes.

[0112] As used herein, MAGE-A2 refers not only to the full-length sequence but also to its variants and fragments. The amino acid sequence of MAGE-A2 (SEQ ID NO:29) is provided in Table 1C (UniProtKB–P43356).

[0113] Table 1. Amino acid sequence of C.MAGE-A2

[0114]

[0115] As used herein, the term "HLA" refers to human leukocyte antigens. In humans, HLA genes encode major histocompatibility complex (MHC) proteins. MHC proteins are expressed on the surface of cells and are involved in the activation of immune responses. HLA class II genes encode MHC class II proteins expressed on the surface of professional antigen-presenting cells (APCs). Non-restricted examples of professional APCs include monocytes, macrophages, dendritic cells (DCs), and B lymphocytes. Some endothelial and epithelial cells also express MHC class II molecules after inflammatory signals are activated. Individuals lacking functional MHC class II molecules are highly susceptible to a range of infectious diseases and typically die at a young age.

[0116] As used herein, "HLA class II molecule" or "MHC class II molecule" refers to the protein product of a wild-type or variant HLA class II gene encoding an MHC class II molecule. Therefore, "HLA class II molecule" and "MHC class II molecule" are used interchangeably in this document. A typical MHC class II molecule contains two protein chains: an α chain and a β chain. Generally, naturally occurring α and β chains each contain a transmembrane domain that anchors the α / β chain to the cell surface, and an extracellular domain that carries antigens and interacts with TCRs and / or CD4 expressed on the cell.

[0117] Both the α and β chains of MHC class II proteins are encoded by the HLA gene complex. The HLA complex is located in the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes with different functions. It is known in the art that the HLA gene complex is highly variable, with more than 20,000 HLA alleles and associated alleles, including more than 250 MHC class II α chain alleles and 5,000 MHC class II β chain alleles, encoding thousands of MHC class II proteins (see, e.g., hla.alleles.org, last accessed May 20, 2019, incorporated herein by reference in its entirety). For example, one such HLA-DP allele, DP4, is the most common allele in many ethnic groups.

[0118] Three loci in the HLA complex encode MHC class II proteins: HLA-DP, HLA-DQ, and HLA-DR. HLA-DO and HLA-DM encode proteins that associate with MHC class II molecules and support their conformation and function.

[0119] When MHC class II molecules complex with antigenic peptides, the 10-30 amino acid-long antigenic peptide binds to a peptide-binding groove and is presented extracellularly to CD4+ cells. Both the α and β chains fold into two separate domains: α-1 and α-2 of the α peptide, and β-1 and β-2 of the β peptide. An open peptide-binding groove, maintaining antigen presentation, is found between the α-1 and β-1 domains. Upon interaction with CD4+ T cells, the MHC class II complex interacts with the T cell receptor (TCR) expressed on the T cell surface. Furthermore, the β chain of the MHC class II molecule interacts with the CD4 weakly interacting receptor (K-K) expressed on the T cell surface. D >2mM). The exemplary CD4 amino acid sequence (UniProt-P01730) is provided in Table 2 (SEQ ID NO:10).

[0120] Table 2. Human CD4 amino acid sequence

[0121]

[0122] The term "autologous" refers to any material derived from the same individual and subsequently reintroduced into that individual. For example, autologous T-cell therapy involves administering T cells isolated from the same individual to a subject. The term "allogeneic" refers to any material derived from one individual and subsequently introduced into another individual of the same species. For example, allogeneic T-cell transplantation involves administering T cells obtained from a donor other than the subject to a subject.

[0123] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors. 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 white blood cell malignancies. In some aspects, the method of the present invention can be used to reduce the size of tumors originating from, for example, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or ocular malignant melanoma, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or ocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, 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), esophageal cancer, small bowel cancer, and endocrine cancer. Systemic cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute or chronic leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, including those induced by asbestos, environmentally induced cancers, other B-cell malignancies, and combinations thereof. Certain cancers may respond to chemotherapy or radiation therapy, or the cancer may be refractory.

[0124] Refractory cancers are those that are not suitable for surgical intervention and that initially do not respond to chemotherapy or radiation therapy, or that become unresponsive over time.

[0125] As used in this article, "anti-tumor effect" refers to an increase in tumor volume, number of tumor cells, proliferation of tumor cells, number of metastases, overall survival or progression-free survival, life expectancy, or improvement in various tumor-related physiological symptoms. Anti-tumor effect can also refer to the prevention of tumor development, such as through vaccines.

[0126] As used herein, the term “progression-free survival” may be abbreviated as PFS, which refers to the time from the date of treatment to the date of disease progression or death from any cause according to the revised IWG criteria for response to malignant lymphoma.

[0127] As used in this article, “disease progression” or “progressive disease” may be abbreviated as PD, referring to the worsening of one or more symptoms associated with a specific disease. For example, disease progression in a subject with cancer may include an increase in the number or size of one or more malignant lesions, tumor metastasis, and death.

[0128] As used in this article, “duration of response” (DOR) refers to the time between a subject’s first objective response and the date of disease progression or death as confirmed by the revised IWG criteria for response to malignant lymphoma.

[0129] The term "overall survival" can be abbreviated as OS, and is defined as the time from the date of treatment to the date of death.

[0130] As used herein, a "cytokine" is a non-antibody protein released by a 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. Cytokines can be expressed endogenously by cells or administered to a subject. Immune cells (including macrophages, B cells, T cells, and mast cells) can release cytokines to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. 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, while pro-inflammatory cytokines promote inflammatory responses. 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)γ. 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)-α, TNF-β, 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).

[0131] Chemokines are a type of cytokine that mediates cellular chemotaxis or directed movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eosinophil chemokine, eosinophil chemokine-3, macrophage-derived chemokines (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), γ-inducible protein 10 (IP-10), and thymus and activation-regulated chemokines (TARC or CCL17).

[0132] Other examples of analytes and cytokines of the present invention include, but are not limited to, chemokine (CC 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), and leukemia inhibitory factor (G-CSF). LIF), oncogene M (OSM), CD154, lymphotoxin (LT)β, 4-1BB ligand (4-1BBL), 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 expression ligand 1 (TALL-1) or TNF-related apoptosis-inducing ligand (TRAIL).

[0133] The term "therapeutic effective amount," "effective dose," "effective amount," or "therapeutic effective dose" refers to any amount of the drug, when used alone or in combination with another therapeutic agent, that demonstrates protection of a subject from disease onset or promotion of disease remission by reducing the severity of disease symptoms, increasing the frequency and duration of asymptomatic periods of disease, or preventing injury or disability caused by the disease. The ability of a therapeutic agent to promote disease remission can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by measuring the activity of the agent in vitro.

[0134] As used herein, the term "infection" refers to any type of invasion of one or more tissues of the body by a foreign substance. The term "infection" includes, but is not limited to, infections caused by viruses (including viroids and prions), bacteria, fungi, parasites, and any combination thereof.

[0135] As used herein, the term “lymphocyte” includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic / cell toxic lymphocyte that represents a major component of the innate immune system. NK cells repel tumor cells and cells infected by viruses. They function through apoptosis, or programmed cell death. They are called “natural killers” because they can kill cells without activation. T cells play a major role in cell-mediated immunity (without antibody involvement). The T cell receptor (TCR) distinguishes T cells from other lymphocyte types. The thymus is a specialized organ of the immune system, primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells (i.e., stem memory T cells), and memory T cells. SCM Cells, like immature 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 exhibit many functional properties unique to memory cells; (ii) central memory T cells CM Cells express L-selectin and CCR7; they secrete IL-2 but not IFNγ or IL-4, and (iii) effector memory T cells. EM B cells, however, do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4, regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and γδ T cells. On the other hand, B cells play a major role in humoral immunity (with antibody involvement). B cells produce antibodies and antigens and act as antigen-presenting cells (APCs), transforming into memory B cells upon activation by antigen-antigen interactions. In mammals, immature B cells form in the bone marrow, from which their name originates.

[0136] The terms “modified” and “mutated” as used herein to refer to a nucleotide or amino acid sequence mean a change in sequence relative to a wild-type sequence or a specific reference sequence. Unless otherwise stated, the terms “modified” and “mutated” do not require steps in the process of preparing a modified or mutated sequence (e.g., a modified β-chain sequence). Rather, these terms indicate a variation in the modified or mutated sequence relative to a reference sequence (e.g., a wild-type sequence). For example, a DPβ chain with a substitution mutation at the position corresponding to amino acid residue 112 of SEQ ID NO: 1 does not require that the wild-type DPβ chain has been physically altered to obtain the DPβ chain; rather, when properly aligned, the DPβ chain contains amino acid residues at the position (residue 112) that differ from the amino acid residues at the corresponding position in the wild-type or reference DPβ chain.

[0137] As used herein, the term "any amino acid" refers to any known amino acid. An amino acid is an organic compound containing (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 and any modifications and variants thereof. There are approximately 500 naturally occurring amino acids, of which 20 are encoded by the genetic code. Amino acids with positively charged side chains include arginine (Arg; R), histidine (His; H), and lysine (Lys; K). Amino acids with negatively charged side chains include aspartic acid (Asp; D) and glutamic acid (Glu; E). Amino acids with polar, uncharged side chains include serine (Ser; S), threonine (Thr; T), glutamine (Gln; Q), and asparagine (Asn; N). Amino acids with hydrophobic side chains include alanine (Ala; A), isoleucine (Ile; I), leucine (Leu; L), methionine (Met; M), phenylalanine (Phe; F), valine (Val; V), tryptophan (Trp; W), and tyrosine (Tyr; Y). Tryptophan (Trp; W), tyrosine (Tyr; Y), and methionine (Met; M) can also be classified as polar and / or amphiphilic amino acids because these amino acids are commonly found on the surface of protein or lipid membranes. Other amino acids include cysteine ​​(Cys; C), selenocysteine ​​(Sec; U), glycine (Gly; G), and proline (Pro; P).

[0138] As used herein, “at the position corresponding to…” is a means of identifying a specific amino acid residue (e.g., a specific amino acid position) in a polynucleotide or a specific nucleic acid (e.g., a specific nucleic acid position) in a polypeptide. The position can be determined by correctly aligning the sequence in question with a reference sequence. Those skilled in the art will readily understand how to align sequences to determine relative positions. For example, various alignment tools are available online, including but not limited to “Clustal Omega Multiple Sequence Alignment” available at www.ebi.ac.uk (last accessed May 25, 2019).

[0139] The terms "genetically engineered" or "engineered" refer to methods of modifying the genome of cells, including but not limited to deleting coding or non-coding regions or portions thereof, or inserting coding regions or portions thereof. In some aspects, the modified cells are lymphocytes, such as CD4-expressing T cells, or modified cells that can be obtained from a patient or donor. Cells can be modified to express exogenous constructs, such as the T-cell receptor (TCR) disclosed herein, which are incorporated into the cell genome. In some aspects, cells are modified to express CD4.

[0140] "Immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including alpha-1, cytokines, and complement) produced by any of these cells or the liver, which cause selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infecting cells or tissues, cancer cells or other abnormal cells, or (in the case of autoimmune or pathological inflammation) normal human cells or tissues in vertebrates.

[0141] The term "immunotherapy" refers to the treatment of a subject who has a disease or is at risk of contracting or relapsing into a disease by means of methods including inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T-cell therapy. T-cell therapy may include adoptive T-cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T-cell transplantation.

[0142] The cells used in the immunotherapy described herein can be derived from any source known in the art. For example, T cells can be differentiated in vitro from hematopoietic stem cell populations, or T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be generated using any number of techniques known to those skilled in the art (such as FICOLL). TM Isolation and / or apheresis are obtained from units of blood collected from the subject. Further methods for isolating T cells for T-cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety. Immunotherapy may also include administering modified cells to the subject, wherein said modified cells express the CD4 and TCR disclosed herein. In some aspects, the modified cells are not T cells.

[0143] As used herein, “patient” includes anyone with cancer (e.g., lymphoma or leukemia). The terms “subject” and “patient” are used interchangeably in this document.

[0144] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can form the sequence of a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers both to short chains, such as those commonly referred to in the art as peptides, oligopeptides, and oligomers, and to longer chains, which are commonly referred to in the art as proteins, among many types. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0145] As used herein, “stimulus” refers to a primary response induced by the binding of a stimulating molecule to its homologous ligand, wherein this binding mediates a signal transduction event. A “stimulating molecule” is a molecule on T cells, such as the T cell receptor (TCR) / CD4 complex, which specifically binds to a homologous stimulating ligand present on antigen-presenting cells. A “stimulating ligand” is a stimulating molecule on T cells that, when present on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.), can specifically bind to the stimulating molecule on T cells, thereby mediating primary T cell responses, including but not limited to activation, initiation of an immune response, and proliferation. Stimulating ligands include, but are not limited to, MHC class II molecules loaded with peptides, anti-CD4 antibodies, hyperagonist anti-CD2 antibodies, hyperagonist anti-CD28 antibodies, and hyperagonist anti-CD3 antibodies.

[0146] The terms “conditioning” and “preconditioning” are used interchangeably herein and refer to preparing a patient requiring T-cell therapy for the appropriate condition. Conditioning, as used herein, includes, but is not limited to, reducing the number of endogenous lymphocytes prior to T-cell therapy, removing cytokine deposition, increasing serum levels of one or more homeostatic cytokines or pro-inflammatory factors, enhancing the effector function of T cells administered after conditioning, enhancing antigen-presenting cell activation and / or availability, or any combination thereof. In one aspect, “conditioning” includes increasing serum levels of one or more of the following cytokines, such as interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-10 (IL-10), interleukin-5 (IL-5), γ-inducible protein 10 (IP-10), interleukin-8 (IL-8), monocyte chemoattractant 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" includes increasing serum levels of IL-7, IL-15, IP-10, MCP-1, PLGF, CRP, or any combination thereof.

[0147] "Treatment" in a subject refers to any type of intervention or treatment administered to the subject, or the administration of an active agent, with the aim of reversing, reducing, improving, suppressing, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or conditions, or biochemical indicators related to the disease. In one aspect, "treatment" includes partial remission. In another aspect, "treatment" includes complete remission.

[0148] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article "a / an" should be understood to mean "one or more / a" of any of the described or enumerated components.

[0149] The terms “about” or “substantially comprise” refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, in part depending on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “substantially comprise” may mean within one or more standard deviations according to practice in the art. Alternatively, “about” or “substantially comprise” may refer to a range up to 10% (i.e., ±10%). For example, about 3 mg may include any amount between 2.7 mg and 3.3 mg (10%). Furthermore, particularly with respect to biological systems or processes, these terms may refer to values ​​up to an order of magnitude or up to five times the value. When a particular value or composition is provided in this application and claims, unless otherwise stated, it should be assumed that the meaning of “about” or “substantially comprise” is within an acceptable margin of error for that particular value or composition.

[0150] As stated herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range, and, where appropriate, to include fractions thereof (such as one-tenth and one-hundredth of an integer).

[0151] Various aspects of the invention are described in more detail in the following sections.

[0152] II. The method disclosed herein

[0153] This disclosure relates to a method for identifying MHC class II specific TCRs, the method comprising contacting a T cell with a complex comprising (i) an HLA class II molecule having enhanced CD4 binding and (ii) a peptide (e.g., an epitope). In some aspects, the T cell expresses CD4. In some aspects, the T cell expresses one or more TCRs. In some aspects, the MHC class II specific TCR specifically binds to the complex comprising an MHC class II molecule and a peptide.

[0154] In some aspects, MHC class II molecules comprise an α chain and a β chain, wherein the α chain, the β chain, or both the α chain and the β chain contain amino acid sequences with one or more mutations relative to the wild-type α chain and / or β chain of the MHC class II molecule. In some aspects, the α chain contains amino acid sequences with one or more mutations relative to the wild-type α chain of the MHC class II molecule. In some aspects, the α chain contains amino acid sequences with one or more mutations relative to the wild-type α chain of the MHC class II molecule, and the β chain contains amino acid sequences with one or more mutations relative to the wild-type β chain of the MHC class II molecule.

[0155] In some aspects, one or more mutations include substitution mutations. In some aspects, one or more mutations include deletion mutations. In some aspects, one or more mutations include insertion mutations. In some aspects, one or more mutations include replacing a single amino acid with one or more heterologous amino acids. In some aspects, one or more mutations include replacing a single amino acid with a different amino acid. In some aspects, one or more mutations include replacing a single amino acid with two, three, four, five, or more than five different amino acids.

[0156] In some respects, MHC class II molecules are dimers. In some respects, MHC class II molecules are trimers. In some respects, MHC class II molecules are tetramers.

[0157] Certain aspects of this disclosure relate to methods for enriching target T cell populations obtained from human subjects. In some aspects, the method includes contacting T cells with HLA class II molecules disclosed herein. In some aspects, the method includes contacting T cells with cells disclosed herein (e.g., APCs). In some aspects, after contact, the enriched T cell population contains a greater number of T cells capable of binding HLA class II molecules compared to the number of T cells capable of binding HLA class II molecules before contact.

[0158] Some aspects of this disclosure relate to methods for selecting T cells capable of targeting diseased cells (e.g., tumor cells). In some aspects, the method includes contacting an isolated population of T cells in vitro with a complex comprising a fragment of an MHC class II molecule and a polypeptide disclosed herein, said polypeptide being, for example, an antigen expressed by diseased cells, such as a tumor-expressed polypeptide, or, for example, an epitope.

[0159] In some respects, the T cells used in the methods disclosed herein are obtained from human subjects. The T cells obtained from human subjects can be any T cells disclosed herein. In some respects, the T cells obtained from human subjects are tumor-infiltrating lymphocytes (TILs).

[0160] In some aspects, the method further includes selecting T cells that bind to MHC class II molecules. In some aspects, the method further includes administering enriched T cells to a human subject. In some aspects, the subject undergoes preconditioning prior to receiving T cells as described herein.

[0161] In some aspects, the method further includes isolating a TCR that binds to MHC class II molecules. In some aspects, the method further includes sequencing the TCR. In some aspects, the method further includes cloning the TCR. In some aspects, the method further includes recombinantly expressing the TCR or a modified variant thereof in a host cell. In some aspects, the host cell is an immune cell, such as a T cell. In some aspects, the method further includes administering the host cell to a subject. In some aspects, the subject has cancer, and the host cell containing the TCR treats the subject's cancer.

[0162] Class II.A. MHC II molecules

[0163] The human leukocyte antigen (HLA) system (the major histocompatibility complex [MHC] in humans) is an important component of the immune system and is controlled by genes located on chromosome 6. It encodes cell surface molecules on T cells that specifically present antigenic peptides to the TCR. (See also Overview of the Immune System). Antigen-presenting MHC molecules are divided into two main classes: class I MHC molecules and class II MHC molecules.

[0164] Class II MHC molecules exist as transmembrane glycoproteins on the surface of professional antigen-presenting cells (APCs). A complete class II molecule consists of an α-chain and a β-chain. Three loci in the HLA complex encode MHC class II proteins: HLA-DP, HLA-DQ, and HLA-DR. T cells expressing CD4 molecules respond to class II MHC molecules. These lymphocytes typically have effector and helper functions, activating responses to eliminate intracellular pathogens from their own cells or to destroy extracellular parasites and assist other T cells, such as CD8 T cells. Because only professional APCs express class II MHC molecules, only these cells present antigens to CD4 T cells (CD4 binds to the non-polymorphic portions of the α-2 and β-2 domains of the α and β chains of the class II MHC molecules, respectively).

[0165] In some respects, the HLA class II α and β chains are selected from the HLA-DP, HLA-DQ, and HLA-DR alleles. In some respects, the HLA class II β chain is an HLA-DP allele. In some respects, the HLA class II α chain is an HLA-DP allele. In some respects, the HLA class II β chain is an HLA-DQ allele. In some respects, the HLA class II α chain is an HLA-DR allele. In some respects, the HLA class II α chain is an HLA-DR allele.

[0166] II.A.1. HLA-DP molecule

[0167] Many HLA-DP alleles are known in the art, and any known allele can be used in the methods disclosed herein. Examples of HLA-DP α-chain and β-chain alleles are shown in Table 3. An updated list of HLA alleles is available at hla.alleles.org / (last accessed February 27, 2019).

[0168] Table 3: Amino acid and nucleotide sequences of the DPβ and α chains.

[0169]

[0170]

[0171]

[0172]

[0173] II.A.1.a.HLA-DPβ chain

[0174] In some aspects, HLA class II molecules comprise a DPβ chain, wherein the DPβ chain comprises an amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1. Any amino acid other than leucine may be present at position 112 of amino acid residue SEQ ID NO:1. In some aspects, the amino acid other than leucine is an amino acid containing a hydrophobic side chain. In some aspects, the amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1 is an amino acid selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1 is alanine. In some aspects, the amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1 is valine. In some aspects, the amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1 is isoleucine. In some aspects, the amino acid at position 112 of amino acid residue SEQ ID NO:1, other than leucine, is methionine. In some aspects, the amino acid at position 112 of amino acid residue SEQ ID NO:1, other than leucine, is phenylalanine. In some aspects, the amino acid at position 112 of amino acid residue SEQ ID NO:1, other than leucine, is tyrosine. In some aspects, the amino acid at position 112 of amino acid residue SEQ ID NO:1, other than leucine, is tryptophan.

[0175] In some embodiments, the amino acid at position 112 corresponding to amino acid residue 112 of SEQ ID NO:1, excluding leucine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a hydrophobic side chain. In some aspects, the amino acid at position 112 corresponding to amino acid residue 112 of SEQ ID NO:1, excluding leucine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a hydrophobic side chain.

[0176] In some aspects, HLA class II molecules comprise a DPβ chain, wherein the DPβ chain comprises an amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1. Any amino acid other than valine may be present at position 141 of amino acid residue SEQ ID NO:1. In some aspects, the amino acid other than valine is an amino acid containing a hydrophobic side chain. In some aspects, the amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1 is an amino acid selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1 is alanine. In some aspects, the amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1 is isoleucine. In some aspects, the amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1 is leucine. In some aspects, the amino acid at position 141 of amino acid residue SEQ ID NO:1, other than valine, is methionine. In some aspects, the amino acid at position 141 of amino acid residue SEQ ID NO:1, other than valine, is phenylalanine. In some aspects, the amino acid at position 141 of amino acid residue SEQ ID NO:1, other than valine, is tyrosine. In some aspects, the amino acid at position 141 of amino acid residue SEQ ID NO:1, other than valine, is tryptophan.

[0177] In some aspects, the amino acid at position 141 corresponding to amino acid residue 141 of SEQ ID NO:1, excluding valine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a hydrophobic side chain. In some aspects, the amino acid at position 141 corresponding to amino acid residue 141 of SEQ ID NO:1, excluding valine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a hydrophobic side chain.

[0178] In some aspects of this disclosure, MHC class II molecules comprise a DPβ chain containing more than one substitution mutation relative to the wild-type DPβ chain. In some aspects, the DPβ chain contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mutations relative to the wild-type DPβ chain.

[0179] In some aspects, the DPβ chain comprises an amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1 and an amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1. In some aspects, (i) the amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1, (ii) the amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1, or each of the amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1 and the amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1 is an amino acid containing a hydrophobic side chain. In some respects, (i) the amino acid other than leucine at the position corresponding to amino acid residue 112 of SEQ ID NO:1 is selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine and tryptophan; and (ii) the amino acid other than valine at the position corresponding to amino acid residue 141 of SEQ ID NO:1 is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan.

[0180] In some aspects, (i) the amino acid at position 112 of amino acid residue SEQ ID NO:1, excluding leucine, is tryptophan; and (ii) the amino acid at position 141 of amino acid residue SEQ ID NO:1, excluding valine, is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, (i) the amino acid at position 112 of amino acid residue SEQ ID NO:1, excluding leucine, is selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan; and (ii) the amino acid at position 141 of amino acid residue SEQ ID NO:1, excluding valine, is methionine. In some aspects, (i) the amino acid at position 112 of amino acid residue SEQ ID NO:1, excluding leucine, is tryptophan; and (ii) the amino acid at position 141 of amino acid residue SEQ ID NO:1, excluding valine, is methionine.

[0181] In some aspects, the DPβ chain further includes an amino acid other than valine at position 114 of amino acid residue SEQ ID NO:1. In some aspects, the amino acid other than valine at position 114 of amino acid residue SEQ ID NO:1 is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than valine at position 114 of amino acid residue SEQ ID NO:1 is methionine.

[0182] In some aspects, the DPβ chain further includes an amino acid other than methionine at position 158 of amino acid residue SEQ ID NO:1. In some aspects, the amino acid other than methionine at position 158 of amino acid residue SEQ ID NO:1 is selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than methionine at position 158 of amino acid residue SEQ ID NO:1 is isoleucine.

[0183] In some aspects, the DPβ chain comprises (i) amino acids other than leucine at position 112 corresponding to amino acid residue SEQ ID NO:1, and (ii) amino acids other than valine at position 114 corresponding to amino acid residue SEQ ID NO:1. In some aspects, the DPβ chain comprises (i) amino acids other than leucine at position 112 corresponding to amino acid residue SEQ ID NO:1, and (ii) amino acids other than methionine at position 158 corresponding to amino acid residue SEQ ID NO:1.

[0184] In some aspects, the DPβ chain comprises (i) tryptophan at position 112 of amino acid residue SEQ ID NO:1, and (ii) methionine at position 114 of amino acid residue SEQ ID NO:1. In some aspects, the DPβ chain comprises (i) tryptophan at position 112 of amino acid residue SEQ ID NO:1, and (ii) isoleucine at position 158 of amino acid residue SEQ ID NO:1.

[0185] In some aspects, the DPβ chain comprises (i) amino acids other than valine at position 141 corresponding to amino acid residue 1 of SEQ ID NO:1, and (ii) amino acids other than valine at position 114 corresponding to amino acid residue 1 of SEQ ID NO:1. In some aspects, the DPβ chain comprises (i) amino acids other than valine at position 141 corresponding to amino acid residue 1 of SEQ ID NO:1, and (ii) amino acids other than methionine at position 158 corresponding to amino acid residue 1 of SEQ ID NO:1.

[0186] In some aspects, the DPβ chain comprises (i) methionine at position 141 corresponding to amino acid residue SEQ ID NO:1, and (ii) an amino acid other than valine at position 114 corresponding to amino acid residue SEQ ID NO:1. In some aspects, the DPβ chain comprises (i) methionine at position 141 corresponding to amino acid residue SEQ ID NO:1, and (ii) isoleucine at position 158 corresponding to amino acid residue SEQ ID NO:1.

[0187] In some respects, the DPβ chain comprises (i) an amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1, (ii) an amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1, (iii) an amino acid other than valine at position 114 of amino acid residue SEQ ID NO:1, and (iv) an amino acid other than methionine at position 158 of amino acid residue SEQ ID NO:1.

[0188] In some respects, the DPβ chain contains (i) tryptophan at position 112 of amino acid residue SEQ ID NO:1, (ii) methionine at position 141 of amino acid residue SEQ ID NO:1, (iii) methionine at position 114 of amino acid residue SEQ ID NO:1, and (iv) isoleucine at position 158 of amino acid residue SEQ ID NO:1.

[0189] In some aspects, the DPβ chain contains valine at position 114 of amino acid residue SEQ ID NO:1. In some aspects, the DPβ chain contains methionine at position 158 of amino acid residue SEQ ID NO:1. In some aspects, the DPβ chain contains (i) an amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1, (ii) an amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1, and (iii) valine at position 114 of amino acid residue SEQ ID NO:1. In some aspects, the DPβ chain contains (i) an amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1, (ii) an amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1, and (iii) methionine at position 158 of amino acid residue SEQ ID NO:1. In some respects, the DPβ chain comprises (i) an amino acid other than leucine at position 112 of amino acid residue SEQ ID NO:1, (ii) an amino acid other than valine at position 141 of amino acid residue SEQ ID NO:1, (iii) valine at position 114 of amino acid residue SEQ ID NO:1, and (iv) methionine at position 158 of amino acid residue SEQ ID NO:1.

[0190] In some aspects, the DPβ chain comprises (i) tryptophan at position 112 corresponding to amino acid residue 112 of SEQ ID NO:1, (ii) methionine at position 141 corresponding to amino acid residue 141 of SEQ ID NO:1, and (iii) valine at position 114 corresponding to amino acid residue 114 of SEQ ID NO:1. In some aspects, the DPβ chain comprises (i) tryptophan at position 112 corresponding to amino acid residue 112 of SEQ ID NO:1, (ii) methionine at position 141 corresponding to amino acid residue 141 of SEQ ID NO:1, and (iii) methionine at position 158 corresponding to amino acid residue 158 of SEQ ID NO:1. In some aspects, the DPβ chain comprises (i) tryptophan at position 112 corresponding to amino acid residue 112 of SEQ ID NO:1, (ii) methionine at position 141 corresponding to amino acid residue 141 of SEQ ID NO:1, (iii) valine at position 114 corresponding to amino acid residue 114 of SEQ ID NO:1, and (iv) methionine at position 158 corresponding to amino acid residue 158 of SEQ ID NO:1.

[0191] In some respects, the DPβ chain contains (i) tryptophan at position 112 of amino acid residue SEQ ID NO:1, (ii) methionine at position 141 of amino acid residue SEQ ID NO:1, (iii) methionine at position 114 of amino acid residue SEQ ID NO:1, and (iv) isoleucine at position 158 of amino acid residue SEQ ID NO:1.

[0192] In some respects, the DPβ chain described herein exhibits increased affinity for CD4 protein compared to a reference HLA class II molecule. In some respects, the reference HLA class II molecule is an HLA class II molecule possessing a wild-type DPβ chain. In some respects, the reference HLA class II molecule is an HLA class II molecule possessing a DPβ chain comprising (i) a leucine residue at position 112 corresponding to amino acid residue SEQ ID NO:1 and / or (ii) a valine residue at position 141 corresponding to amino acid residue SEQ ID NO:1.

[0193] In some respects, the increased affinity for CD4 is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 1000 times, at least about 1500 times, at least about 2000 times, at least about 2500 times, at least about 3000 times, at least about 3500 times, at least about 4000 times, at least about 4500 times, or at least about 4000 times.

[0194] In some respects, the increased affinity for CD4 is at least approximately 1.5 to at least approximately 5000 times, 1.5 to at least approximately 4000 times, 1.5 to at least approximately 3000 times, 1.5 to at least approximately 2000 times, 1.5 to at least approximately 1000 times, 10 to at least approximately 5000 times, 10 to at least approximately 4000 times, 10 to at least approximately 3000 times, 10 to at least approximately 2000 times, 10 to at least approximately 1000 times, 10 to at least approximately 900 times, 10 to at least approximately 800 times, 10 to at least approximately 700 times, 10 to at least approximately 600 times, 10 to at least approximately 500 times, and 10 to at least approximately 500 times, respectively. At least 400 times, 10 times to at least 300 times, 10 times to at least 200 times, 10 times to at least 100 times, 100 times to at least 5000 times, 100 times to at least 4000 times, 100 times to at least 3000 times, 100 times to at least 2000 times, 100 times to at least 1000 times, 100 times to at least 900 times, 100 times to at least 800 times, 100 times to at least 700 times, 100 times to at least 600 times, 100 times to at least 500 times, 100 times to at least 400 times, 100 times to at least 300 times, or 100 times to at least 200 times.

[0195] In some respects, the DPβ chain contains alleles selected from the following: 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, DP B1*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, D PB1*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, DP B1*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*9 30. 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, D PB1*946, DPB1*947, DPB1*948, DPB1*949, DPB1*95, DPB1*950, DPB1*951, DPB1*952, DPB The DPβ chain contains alleles of HLA-DPB1*01, HLA-DPB1*02, HLA-DPB1*03, HLA-DPB1*04, HLA-DPB1*05, HLA-DPB1*06, HLA-DPB1*08, and HLA-DPB1*09. In some respects, the DPβ chain contains alleles of HLA-DPB1*04, HLA-DPB1*05, HLA-DPB1*06, HLA-DPB1*08, or HLA-DPB1*09. In some respects, the DPβ chain contains the HLA-DPB1*04 allele. In certain respects, the DPβ chain contains the HLA-DPB1*04:01 allele.

[0196] In some respects, MHC class II molecules contain a DPβ 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 with SEQ ID NO:3, wherein the DPβ chain contains tryptophan at position 112 corresponding to amino acid residue 112 of SEQ ID NO:1, and wherein the DPβ chain contains methionine at position 141 corresponding to amino acid residue 141 of SEQ ID NO:1. In some aspects, MHC class II molecules comprise a DPβ chain containing 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 with SEQ ID NO:3, wherein the DPβ chain comprises (i) tryptophan at position 112 corresponding to amino acid residue 112 of SEQ ID NO:1, (ii) methionine at position 141 corresponding to amino acid residue 141 of SEQ ID NO:1, (iii) valine at position 114 corresponding to amino acid residue 114 of SEQ ID NO:1, and (iv) methionine at position 158 corresponding to amino acid residue 158 of SEQ ID NO:1. In some aspects, MHC class II molecules comprise a DPβ chain containing the amino acid sequence shown in SEQ ID NO:3.

[0197] II.A.1.a.HLA-DPα chain

[0198] In some aspects of this disclosure, MHC class II molecules also comprise an α chain. In some aspects, the α chain is a wild-type α chain. In some aspects, the α chain is a DPα chain. Any DPα chain may be used in the compositions and methods of this disclosure. In some aspects, the DPα chain comprises an HLA-DPA1*01, HLA-DPA1*02, HLA-DPA1*03, or HLA-DPA1*04 allele. In some aspects, the DPα chain comprises an HLA-DPA1*01 allele. In some aspects, the DPα chain comprises an HLA-DPA1*02 allele. In some aspects, the DPα chain comprises an HLA-DPA1*03 allele. In some aspects, the DPα chain comprises an HLA-DPA1*04 allele.

[0199] In some aspects, the DPα 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*02:0 2:02:02, 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*02:0 5. DPA1*02:06, DPA1*02:07:01:01, DPA1*02:07:01:02, DPA1*02:07:01:03, DPA1*02:0 8. 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.

[0200] In some aspects, MHC class II molecules comprise a DPα chain containing 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 with SEQ ID NO:6. In some aspects, MHC class II molecules comprise a DPα chain containing 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 with SEQ ID NO:8. In some aspects, MHC class II molecules comprise a DPα chain containing the amino acid sequence shown in SEQ ID NO:6. In some respects, MHC class II molecules contain a DPα chain containing the amino acid sequence shown in SEQ ID NO:8.

[0201] II.A.2.HLA-DQ molecule

[0202] Many HLA-DQ alleles are known in the art, and any known allele can be used in this disclosure. Examples of HLA-DQ α-chain and β-chain alleles are shown in Table 4. An updated list of HLA alleles is available at hla.alleles.org / (last accessed July 10, 2019).

[0203] Table 4: Amino acid and nucleotide sequences of the DQ β and α chains.

[0204]

[0205]

[0206] II.A.2.a.HLA-DQβ chain

[0207] In some aspects, HLA class II molecules comprise a DQβ chain, wherein the DQβ chain comprises an amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11. Any amino acid other than leucine may be present at position 114 of amino acid residue SEQ ID NO:11. In some aspects, the amino acid other than leucine is an amino acid containing a hydrophobic side chain. In some aspects, the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11 is an amino acid selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11 is alanine. In some aspects, the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11 is valine. In some aspects, the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11 is isoleucine. In some aspects, the amino acid at position 114 of amino acid residue SEQ ID NO:11, other than leucine, is methionine. In some aspects, the amino acid at position 114 of amino acid residue SEQ ID NO:11, other than leucine, is phenylalanine. In some aspects, the amino acid at position 114 of amino acid residue SEQ ID NO:11, other than leucine, is tyrosine. In some aspects, the amino acid at position 114 of amino acid residue SEQ ID NO:11, other than leucine, is tryptophan.

[0208] In some embodiments, the amino acid at position 114 corresponding to amino acid residue 114 of SEQ ID NO:11, excluding leucine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a hydrophobic side chain. In some aspects, the amino acid at position 114 corresponding to amino acid residue 114 of SEQ ID NO:11, excluding leucine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a hydrophobic side chain.

[0209] In some aspects, HLA class II molecules comprise a DQβ chain, wherein the DQβ chain comprises an amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11. Any amino acid other than valine may be present at position 143 of amino acid residue SEQ ID NO:11. In some aspects, the amino acid other than valine is an amino acid containing a hydrophobic side chain. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11 is an amino acid selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11 is alanine. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11 is isoleucine. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11 is leucine. In some aspects, the amino acid at position 143 of amino acid residue SEQ ID NO:11, other than valine, is methionine. In some aspects, the amino acid at position 143 of amino acid residue SEQ ID NO:11, other than valine, is phenylalanine. In some aspects, the amino acid at position 143 of amino acid residue SEQ ID NO:11, other than valine, is tyrosine. In some aspects, the amino acid at position 143 of amino acid residue SEQ ID NO:11, other than valine, is tryptophan.

[0210] In some aspects, the amino acid at position 143 corresponding to amino acid residue SEQ ID NO:11, excluding valine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a hydrophobic side chain. In some aspects, the amino acid at position 143 corresponding to amino acid residue SEQ ID NO:11, excluding valine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a hydrophobic side chain.

[0211] In some aspects, HLA class II molecules comprise a DQβ chain, wherein the DQβ chain comprises an amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11. Any amino acid other than asparagine may be present at position 110 of amino acid residue SEQ ID NO:11. In some aspects, the amino acid other than asparagine is an amino acid comprising a polar, uncharged side chain. In some aspects, the amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11 is an amino acid selected from serine, threonine, and glutamine. In some aspects, the amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11 is serine. In some aspects, the amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11 is threonine. In some aspects, the amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11 is glutamine.

[0212] In some aspects, the amino acid at position 110 of amino acid residue SEQ ID NO:11, excluding asparagine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a polar, uncharged side chain. In some aspects, the amino acid at position 110 of amino acid residue SEQ ID NO:11, excluding asparagine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a polar, uncharged side chain.

[0213] In some aspects, HLA class II molecules comprise a DQβ chain, wherein the DQβ chain comprises an amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11. Any amino acid other than isoleucine may be present at position 116 of amino acid residue SEQ ID NO:11. In some aspects, the amino acid other than isoleucine is an amino acid containing a hydrophobic side chain. In some aspects, the amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11 is an amino acid selected from alanine, valine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11 is alanine. In some aspects, the amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11 is valine. In some aspects, the amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11 is leucine. In some aspects, the amino acid at position 116 of amino acid residue SEQ ID NO:11, excluding isoleucine, is methionine. In some aspects, the amino acid at position 116 of amino acid residue SEQ ID NO:11, excluding isoleucine, is phenylalanine. In some aspects, the amino acid at position 116 of amino acid residue SEQ ID NO:11, excluding isoleucine, is tyrosine. In some aspects, the amino acid at position 116 of amino acid residue SEQ ID NO:11, excluding isoleucine, is tryptophan.

[0214] In some aspects, the amino acid at position 116 corresponding to amino acid residue SEQ ID NO:11, excluding isoleucine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a hydrophobic side chain. In some aspects, the amino acid at position 116 corresponding to amino acid residue SEQ ID NO:11, excluding isoleucine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a hydrophobic side chain.

[0215] In some aspects, HLA class II molecules comprise a DQβ chain, wherein the DQβ chain comprises an amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11. Any amino acid other than serine may be present at position 118 of amino acid residue SEQ ID NO:11. In some aspects, the amino acid other than serine is an amino acid containing a charged side chain. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11 is an amino acid selected from arginine, histidine, and lysine. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11 is arginine. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11 is histidine. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11 is lysine.

[0216] In some aspects, the amino acid at position 118 corresponding to amino acid residue 111, excluding serine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a charged side chain. In some aspects, the amino acid at position 118 corresponding to amino acid residue 111, excluding serine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a charged side chain.

[0217] In some aspects, HLA class II molecules comprise a DQβ chain, wherein the DQβ chain comprises an amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11. Any amino acid other than proline may be present at position 146 of amino acid residue SEQ ID NO:11. In some aspects, the amino acid other than proline is an amino acid comprising a polar, uncharged side chain. In some aspects, the amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11 is an amino acid selected from serine, threonine, asparagine, and glutamine. In some aspects, the amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11 is serine. In some aspects, the amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11 is threonine. In some aspects, the amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11 is asparagine. In some respects, the amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11 is glutamine.

[0218] In some aspects, the amino acid at position 146 corresponding to amino acid residue SEQ ID NO:11, excluding proline, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a polar, uncharged side chain. In some aspects, the amino acid at position 146 corresponding to amino acid residue SEQ ID NO:11, excluding proline, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a polar, uncharged side chain.

[0219] In some aspects of this disclosure, MHC class II molecules comprise a DQβ chain containing more than one substitution mutation relative to the wild-type DQβ chain. In some aspects, the DQβ chain contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mutations relative to the wild-type DQβ chain.

[0220] In some aspects, the DQβ chain comprises an amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11 and an amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11. In some aspects, the DQβ chain comprises an amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11; an amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11; and at least three of the following: (i) an amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11, (ii) an amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11, (iii) an amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11, and (iv) an amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11.

[0221] In some respects, the DQβ chain comprises (i) an amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11; (ii) an amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11; (iii) an amino acid other than asparagine at position 110 of amino acid residue SEQ ID NO:11; (iv) an amino acid other than isoleucine at position 116 of amino acid residue SEQ ID NO:11; (v) an amino acid other than serine at position 118 of amino acid residue SEQ ID NO:11; and (vi) an amino acid other than proline at position 146 of amino acid residue SEQ ID NO:11.

[0222] In some respects, (i) the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11, (ii) the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11, or each of the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:11 and the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:11 is an amino acid containing a hydrophobic side chain.

[0223] In some respects, (i) the amino acid at position 114 of amino acid residue SEQ ID NO:11, excluding leucine, is selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan; (ii) the amino acid at position 143 of amino acid residue SEQ ID NO:11, excluding valine, is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; (iii) the amino acid at position 110 of amino acid residue SEQ ID NO:11, excluding asparagine, is selected from serine, threonine, and glutamine; (iv) the amino acid at position 116 of amino acid residue SEQ ID NO:11, excluding isoleucine, is selected from alanine, valine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; (v) the amino acid at position 118 of amino acid residue SEQ ID NO:11, excluding serine, is selected from arginine, histidine, and lysine; and (vi) the amino acid at position 143 of amino acid residue SEQ ID NO:11, excluding valine, is selected from alanine, valine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; (iv) the amino acid at position 116 of amino acid residue SEQ ID NO:11, excluding isoleucine, is selected from alanine, valine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; (v) the amino acid at position 118 of amino acid residue SEQ ID NO:11, excluding serine, is selected from arginine, histidine, and lysine; and (vi) the amino acid at position 143 of amino acid residue SEQ The amino acid at position 146 of amino acid residue NO:11, excluding proline, is selected from serine, threonine, asparagine, and glutamine.

[0224] In some aspects, (i) the amino acid at position 114 of amino acid residue SEQ ID NO:11, excluding leucine, is tryptophan; and (ii) the amino acid at position 143 of amino acid residue SEQ ID NO:11, excluding valine, is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, (i) the amino acid at position 114 of amino acid residue SEQ ID NO:11, excluding leucine, is selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan; and (ii) the amino acid at position 143 of amino acid residue SEQ ID NO:11, excluding valine, is methionine. In some aspects, (i) the amino acid at position 114 of amino acid residue SEQ ID NO:11, excluding leucine, is tryptophan; and (ii) the amino acid at position 143 of amino acid residue SEQ ID NO:11, excluding valine, is methionine. In some respects, (i) the amino acid at position 114 of amino acid residue SEQ ID NO:11, excluding leucine, is tryptophan; (ii) the amino acid at position 143 of amino acid residue SEQ ID NO:11, excluding valine, is methionine; (iii) the amino acid at position 110 of amino acid residue SEQ ID NO:11, excluding asparagine, is glutamine; (iv) the amino acid at position 116 of amino acid residue SEQ ID NO:11, excluding isoleucine, is valine; (v) the amino acid at position 118 of amino acid residue SEQ ID NO:11, excluding serine, is histidine; and (vi) the amino acid at position 146 of amino acid residue SEQ ID NO:11, excluding proline, is glutamine.

[0225] In some respects, the DQβ chain described herein exhibits increased affinity for CD4 protein compared to a reference HLA class II molecule. In some respects, the reference HLA class II molecule is an HLA class II molecule possessing a wild-type DQβ chain. In some respects, the reference HLA class II molecule is an HLA class II molecule possessing a DQβ chain comprising (i) a leucine residue at position 114 corresponding to amino acid residue SEQ ID NO: 11 and / or (ii) a valine residue at position 143 corresponding to amino acid residue SEQ ID NO: 11. In some respects, the reference HLA class II molecule is an HLA class II molecule having a DQβ chain comprising (i) leucine at position 114 of amino acid residue SEQ ID NO:11, (ii) valine at position 143 of amino acid residue SEQ ID NO:11, (iii) asparagine at position 110 of amino acid residue SEQ ID NO:11, (iv) isoleucine at position 116 of amino acid residue SEQ ID NO:11, (iii) serine at position 118 of amino acid residue SEQ ID NO:11, and / or (iv) proline at position 146 of amino acid residue SEQ ID NO:11.

[0226] In some respects, the increased affinity for CD4 is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 1000 times, at least about 1500 times, at least about 2000 times, at least about 2500 times, at least about 3000 times, at least about 3500 times, at least about 4000 times, at least about 4500 times, or at least about 4000 times.

[0227] In some respects, the increased affinity for CD4 is at least approximately 1.5 to at least approximately 5000 times, 1.5 to at least approximately 4000 times, 1.5 to at least approximately 3000 times, 1.5 to at least approximately 2000 times, 1.5 to at least approximately 1000 times, 10 to at least approximately 5000 times, 10 to at least approximately 4000 times, 10 to at least approximately 3000 times, 10 to at least approximately 2000 times, 10 to at least approximately 1000 times, 10 to at least approximately 900 times, 10 to at least approximately 800 times, 10 to at least approximately 700 times, 10 to at least approximately 600 times, 10 to at least approximately 500 times, and 10 to at least approximately 500 times, respectively. At least 400 times, 10 times to at least 300 times, 10 times to at least 200 times, 10 times to at least 100 times, 100 times to at least 5000 times, 100 times to at least 4000 times, 100 times to at least 3000 times, 100 times to at least 2000 times, 100 times to at least 1000 times, 100 times to at least 900 times, 100 times to at least 800 times, 100 times to at least 700 times, 100 times to at least 600 times, 100 times to at least 500 times, 100 times to at least 400 times, 100 times to at least 300 times, or 100 times to at least 200 times.

[0228] In some respects, the DQβ chain contains alleles selected from the following: HLA-DQB1*02, HLA-DQB1*03, HLA-DQB1*04, HLA-DQB1*05, and HLA-DQB1*06. In some respects, the DQβ chain contains the HLA-DQB1*05 allele. In certain respects, the DQβ chain contains the HLA-DQB1*05:01 allele.

[0229] In some respects, the DQβ chain contains alleles selected from the following: 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, DQB 1*02:01:17, DQB1*02:01:18, DQB1*02:01:19, DQB1*02:01:20, DQB1*02:0 1:21, DQB1*02:01:22, DQB1*02:01:23, DQB1*02:01:24, DQB1*02:01:25, D QB1*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*02:0 5. 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, DQB 1*02:104, DQB1*02:105, DQB1*02:106, DQB1*02:107, DQB1*02:108, DQB1*0 2:109, DQB1*02:11, DQB1*02:110, DQB1*02:111, DQB1*02:112, DQB1*02:1 13. 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、D QB1*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: 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: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.

[0230] In some respects, MHC class II molecules comprise a DQβ chain containing 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 with SEQ ID NO:13, wherein the DQβ chain contains tryptophan at position 114 corresponding to amino acid residue 114 of SEQ ID NO:11, and wherein the DQβ chain contains methionine at position 143 corresponding to amino acid residue 143 of SEQ ID NO:11. In some respects, MHC class II molecules comprise a DQβ chain containing 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 with SEQ ID NO:13, wherein the DQβ chain comprises (i) tryptophan at position 114 corresponding to amino acid residue 114 of SEQ ID NO:11, (ii) methionine at position 143 corresponding to amino acid residue 143 of SEQ ID NO:11, (iii) glutamine at position 110 corresponding to amino acid residue 110 of SEQ ID NO:11, (iv) valine at position 116 corresponding to amino acid residue 116 of SEQ ID NO:11, (v) histidine at position 118 corresponding to amino acid residue 118 of SEQ ID NO:11, and (vi) glutamine at position 146 corresponding to amino acid residue 146 of SEQ ID NO:11. In some respects, MHC class II molecules contain a DQβ chain, which contains the amino acid sequence shown in SEQ ID NO:13.

[0231] II.A.2.b. HLA-DQα chain

[0232] In some aspects of this disclosure, MHC class II molecules also comprise an α chain. In some aspects, the α chain is a wild-type α chain. In some aspects, the α chain is a DQα chain. Any DQα chain may be used in the compositions and methods of this disclosure. In some aspects, the DQα chain 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 DQα chain comprises an HLA-DQA1*01 allele. In some aspects, the DQα chain comprises an HLA-DQA1*02 allele. In some aspects, the DQα chain comprises an HLA-DQA1*03 allele. In some aspects, the DQα chain comprises an HLA-DQA1*04 allele. In some respects, the DQα chain contains the HLA-DQA1*05 allele. In other respects, the DQα chain contains the HLA-DQA1*06 allele.

[0233] In some respects, the DQα 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, and 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*01:03:0 1:03. 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*01:04:01:02, 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*01:0 8. 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*03:03:04、DQA1*03:03:05、DQA1*03:03:01:07 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*0 5:01:01:01、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*0 5:05:01:05、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.

[0234] In some aspects, MHC class II molecules comprise a DQα chain containing 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 with SEQ ID NO:16. In some aspects, MHC class II molecules comprise a DQα chain containing 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 with SEQ ID NO:18. In some aspects, MHC class II molecules comprise a DQα chain containing the amino acid sequence shown in SEQ ID NO:16. In some respects, MHC class II molecules contain a DQα chain, which contains the amino acid sequence shown in SEQ ID NO:18.

[0235] II.A.3. HLA-DR molecule

[0236] Many HLA-DR alleles are known in the art, and any known allele can be used in this disclosure. Examples of HLA-DR α-chain and β-chain alleles are shown in Table 5. An updated list of HLA alleles is available at hla.alleles.org / (last accessed July 10, 2019).

[0237] Table 5: Amino acid and nucleotide sequences of the DRβ and α chains.

[0238]

[0239]

[0240]

[0241]

[0242] II.A.3a.HLA-DRβ chain

[0243] In some aspects, HLA class II molecules comprise a DRβ chain, wherein the DRβ chain comprises an amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19. Any amino acid other than leucine may be present at position 114 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than leucine is an amino acid containing a hydrophobic side chain. In some aspects, the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19 is an amino acid selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19 is alanine. In some aspects, the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19 is valine. In some aspects, the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19 is isoleucine. In some aspects, the amino acid at position 114 of amino acid residue SEQ ID NO:19, other than leucine, is methionine. In some aspects, the amino acid at position 114 of amino acid residue SEQ ID NO:19, other than leucine, is phenylalanine. In some aspects, the amino acid at position 114 of amino acid residue SEQ ID NO:19, other than leucine, is tyrosine. In some aspects, the amino acid at position 114 of amino acid residue SEQ ID NO:19, other than leucine, is tryptophan.

[0244] In some embodiments, the amino acid at position 114 corresponding to amino acid residue 119, excluding leucine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a hydrophobic side chain. In some aspects, the amino acid at position 114 corresponding to amino acid residue 119, excluding leucine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a hydrophobic side chain.

[0245] In some aspects, HLA class II molecules comprise a DRβ chain, wherein the DRβ chain comprises an amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19. Any amino acid other than valine may be present at position 143 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than valine is an amino acid containing a hydrophobic side chain. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19 is an amino acid selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19 is alanine. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19 is isoleucine. In some aspects, the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19 is leucine. In some aspects, the amino acid at position 143 of amino acid residue SEQ ID NO:19, other than valine, is methionine. In some aspects, the amino acid at position 143 of amino acid residue SEQ ID NO:19, other than valine, is phenylalanine. In some aspects, the amino acid at position 143 of amino acid residue SEQ ID NO:19, other than valine, is tyrosine. In some aspects, the amino acid at position 143 of amino acid residue SEQ ID NO:19, other than valine, is tryptophan.

[0246] In some aspects, the amino acid at position 143 corresponding to amino acid residue SEQ ID NO:19, excluding valine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a hydrophobic side chain. In some aspects, the amino acid at position 143 corresponding to amino acid residue SEQ ID NO:19, excluding valine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a hydrophobic side chain.

[0247] In some aspects, HLA class II molecules comprise a DRβ chain, wherein the DRβ chain comprises an amino acid other than serine at position 118 of amino acid residue SEQ ID NO:19. Any amino acid other than serine may be present at position 118 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than serine is an amino acid containing a charged side chain. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:19 is an amino acid selected from arginine, histidine, and lysine. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:19 is arginine. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:19 is histidine. In some aspects, the amino acid other than serine at position 118 of amino acid residue SEQ ID NO:19 is lysine.

[0248] In some aspects, the amino acid at position 118 corresponding to amino acid residue SEQ ID NO:19, excluding serine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a charged side chain. In some aspects, the amino acid at position 118 corresponding to amino acid residue SEQ ID NO:19, excluding serine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a charged side chain.

[0249] In some aspects, HLA class II molecules contain a DRβ chain, wherein the DRβ chain contains an amino acid other than threonine at position 157 of amino acid residue SEQ ID NO:19. Any amino acid other than threonine may be present at position 157 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than threonine is an amino acid containing a hydrophobic side chain. In some aspects, the amino acid other than threonine at position 157 of amino acid residue SEQ ID NO:19 is an amino acid selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than threonine at position 157 of amino acid residue SEQ ID NO:19 is alanine. In some aspects, the amino acid other than threonine at position 157 of amino acid residue SEQ ID NO:19 is valine. In some aspects, the amino acid other than threonine at position 157 of amino acid residue SEQ ID NO:19 is isoleucine. In some aspects, the amino acid at position 157 of amino acid residue SEQ ID NO:19, other than threonine, is leucine. In some aspects, the amino acid at position 157 of amino acid residue SEQ ID NO:19, other than threonine, is methionine. In some aspects, the amino acid at position 157 of amino acid residue SEQ ID NO:19, other than threonine, is phenylalanine. In some aspects, the amino acid at position 157 of amino acid residue SEQ ID NO:19, other than threonine, is tyrosine. In some aspects, the amino acid at position 157 of amino acid residue SEQ ID NO:19, other than threonine, is tryptophan.

[0250] In some aspects, the amino acid at position 157 of amino acid residue SEQ ID NO:19, excluding threonine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a hydrophobic side chain. In some aspects, the amino acid at position 157 of amino acid residue SEQ ID NO:19, excluding threonine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a hydrophobic side chain.

[0251] In some aspects, HLA class II molecules contain a DRβ chain, wherein the DRβ chain contains an amino acid other than lysine at position 139 of amino acid residue SEQ ID NO:19. Any amino acid other than lysine may be present at position 139 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than lysine is an amino acid containing a polar, uncharged side chain. In some aspects, the amino acid other than lysine at position 139 of amino acid residue SEQ ID NO:19 is an amino acid selected from serine, threonine, and glutamine. In some aspects, the amino acid other than lysine at position 139 of amino acid residue SEQ ID NO:19 is serine. In some aspects, the amino acid other than lysine at position 139 of amino acid residue SEQ ID NO:19 is threonine. In some aspects, the amino acid other than lysine at position 139 of amino acid residue SEQ ID NO:19 is glutamine.

[0252] In some aspects, the amino acid at position 139 corresponding to amino acid residue SEQ ID NO:19, excluding lysine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a polar, uncharged side chain. In some aspects, the amino acid at position 139 corresponding to amino acid residue SEQ ID NO:19, excluding lysine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a polar, uncharged side chain.

[0253] In some aspects, HLA class II molecules contain a DRβ chain, wherein the DRβ chain contains an amino acid other than glycine at position 146 of amino acid residue SEQ ID NO:19. Any amino acid other than glycine may be present at position 146 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than glycine is an amino acid containing a polar, uncharged side chain. In some aspects, the amino acid other than glycine at position 146 of amino acid residue SEQ ID NO:19 is an amino acid selected from serine, asparagine, threonine, and glutamine. In some aspects, the amino acid other than glycine at position 146 of amino acid residue SEQ ID NO:19 is serine. In some aspects, the amino acid other than glycine at position 146 of amino acid residue SEQ ID NO:19 is asparagine. In some aspects, the amino acid other than glycine at position 146 of amino acid residue SEQ ID NO:19 is threonine. In some respects, the amino acid other than glycine at position 146 of amino acid residue SEQ ID NO:19 is glutamine.

[0254] In some aspects, the amino acid at position 146 corresponding to amino acid residue SEQ ID NO:19, excluding glycine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a polar, uncharged side chain. In some aspects, the amino acid at position 146 corresponding to amino acid residue SEQ ID NO:19, excluding glycine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a polar, uncharged side chain.

[0255] In some aspects, HLA class II molecules contain a DRβ chain, wherein the DRβ chain contains an amino acid other than threonine at position 163 of amino acid residue SEQ ID NO:19. Any amino acid other than threonine may be present at position 163 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than threonine is an amino acid containing a hydrophobic side chain. In some aspects, the amino acid other than threonine at position 163 of amino acid residue SEQ ID NO:19 is an amino acid selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some aspects, the amino acid other than threonine at position 163 of amino acid residue SEQ ID NO:19 is alanine. In some aspects, the amino acid other than threonine at position 163 of amino acid residue SEQ ID NO:19 is valine. In some aspects, the amino acid other than threonine at position 163 of amino acid residue SEQ ID NO:19 is isoleucine. In some aspects, the amino acid at position 163 of amino acid residue SEQ ID NO:19, other than threonine, is leucine. In some aspects, the amino acid at position 163 of amino acid residue SEQ ID NO:19, other than threonine, is methionine. In some aspects, the amino acid at position 163 of amino acid residue SEQ ID NO:19, other than threonine, is phenylalanine. In some aspects, the amino acid at position 163 of amino acid residue SEQ ID NO:19, other than threonine, is tyrosine. In some aspects, the amino acid at position 163 of amino acid residue SEQ ID NO:19, other than threonine, is tryptophan.

[0256] In some aspects, the amino acid at position 163 corresponding to amino acid residue SEQ ID NO:19, excluding threonine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a hydrophobic side chain. In some aspects, the amino acid at position 163 corresponding to amino acid residue SEQ ID NO:19, excluding threonine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a hydrophobic side chain.

[0257] In some aspects, HLA class II molecules contain a DRβ chain, wherein the DRβ chain contains an amino acid other than valine at position 164 of amino acid residue SEQ ID NO:19. Any amino acid other than valine may be present at position 164 of amino acid residue SEQ ID NO:19. In some aspects, the amino acid other than valine is an amino acid containing a polar, uncharged side chain. In some aspects, the amino acid other than valine at position 164 of amino acid residue SEQ ID NO:19 is an amino acid selected from serine, asparagine, threonine, and glutamine. In some aspects, the amino acid other than valine at position 164 of amino acid residue SEQ ID NO:19 is serine. In some aspects, the amino acid other than valine at position 164 of amino acid residue SEQ ID NO:19 is asparagine. In some aspects, the amino acid other than valine at position 164 of amino acid residue SEQ ID NO:19 is threonine. In some respects, the amino acid other than valine at position 164 of amino acid residue SEQ ID NO:19 is glutamine.

[0258] In some aspects, the amino acid at position 164 corresponding to amino acid residue SEQ ID NO:19, excluding valine, consists of more than one amino acid, for example, two, three, four, five, or more amino acids. In some aspects, at least one of the more than one amino acid contains a polar, uncharged side chain. In some aspects, the amino acid at position 164 corresponding to amino acid residue SEQ ID NO:19, excluding valine, consists of a series, for example, at least two, at least three, at least four, or at least five amino acids, wherein each of said series of amino acids contains a polar, uncharged side chain.

[0259] In some aspects of this disclosure, MHC class II molecules comprise a DRβ chain containing more than one substitution mutation relative to the wild-type DRβ chain. In some aspects, the DRβ chain contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mutations relative to the wild-type DRβ chain.

[0260] In some respects, the DRβ chain contains amino acids other than leucine at position 114 of amino acid residue SEQ ID NO:19 and amino acids other than valine at position 143 of amino acid residue SEQ ID NO:19.

[0261] In some respects, the DRβ chain comprises (a) an amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19, (b) an amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19, (c) an amino acid other than serine at position 118 of amino acid residue SEQ ID NO:19, and (d) an amino acid other than threonine at position 157 of amino acid residue SEQ ID NO:19.

[0262] In some respects, the DRβ chain comprises an amino acid other than leucine at position 114 corresponding to amino acid residue SEQ ID NO:19; an amino acid other than valine at position 143 corresponding to amino acid residue SEQ ID NO:19; and at least two of the following: (i) an amino acid other than serine at position 118 corresponding to amino acid residue SEQ ID NO:19, (ii) an amino acid other than lysine at position 139 corresponding to amino acid residue SEQ ID NO:19, (iii) an amino acid other than glycine at position 146 corresponding to amino acid residue SEQ ID NO:19, (iv) an amino acid other than threonine at position 157 corresponding to amino acid residue SEQ ID NO:19, (v) an amino acid other than threonine at position 163 corresponding to amino acid residue SEQ ID NO:19, and (vi) an amino acid other than valine at position 164 corresponding to amino acid residue SEQ ID NO:19.

[0263] In some respects, the DRβ chain comprises an amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19; an amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19; and at least three of the following: (i) an amino acid other than serine at position 118 of amino acid residue SEQ ID NO:19, (ii) an amino acid other than lysine at position 139 of amino acid residue SEQ ID NO:19, (iii) an amino acid other than glycine at position 146 of amino acid residue SEQ ID NO:19, (iv) an amino acid other than threonine at position 157 of amino acid residue SEQ ID NO:19, (v) an amino acid other than threonine at position 163 of amino acid residue SEQ ID NO:19, and (vi) an amino acid other than valine at position 164 of amino acid residue SEQ ID NO:19.

[0264] In some respects, the DRβ chain comprises an amino acid other than leucine at position 114 corresponding to amino acid residue SEQ ID NO:19; an amino acid other than valine at position 143 corresponding to amino acid residue SEQ ID NO:19; and at least four of the following: (i) an amino acid other than serine at position 118 corresponding to amino acid residue SEQ ID NO:19, (ii) an amino acid other than lysine at position 139 corresponding to amino acid residue SEQ ID NO:19, (iii) an amino acid other than glycine at position 146 corresponding to amino acid residue SEQ ID NO:19, (iv) an amino acid other than threonine at position 157 corresponding to amino acid residue SEQ ID NO:19, (v) an amino acid other than threonine at position 163 corresponding to amino acid residue SEQ ID NO:19, and (vi) an amino acid other than valine at position 164 corresponding to amino acid residue SEQ ID NO:19.

[0265] In some respects, the DRβ chain comprises (a) an amino acid other than leucine at position 114 corresponding to amino acid residue SEQ ID NO:19, (b) an amino acid other than valine at position 143 corresponding to amino acid residue SEQ ID NO:19, (c) an amino acid other than serine at position 118 corresponding to amino acid residue SEQ ID NO:19, and (d) an amino acid other than threonine at position 157 corresponding to amino acid residue SEQ ID NO:19; and at least one of the following: (i) an amino acid other than lysine at position 139 corresponding to amino acid residue SEQ ID NO:19, (ii) an amino acid other than glycine at position 146 corresponding to amino acid residue SEQ ID NO:19, (iii) an amino acid other than threonine at position 163 corresponding to amino acid residue SEQ ID NO:19, and (iv) an amino acid other than valine at position 164 corresponding to amino acid residue SEQ ID NO:19.

[0266] In some respects, the DRβ chain comprises (a) an amino acid other than leucine at position 114 corresponding to amino acid residue SEQ ID NO:19, (b) an amino acid other than valine at position 143 corresponding to amino acid residue SEQ ID NO:19, (c) an amino acid other than serine at position 118 corresponding to amino acid residue SEQ ID NO:19, and (d) an amino acid other than threonine at position 157 corresponding to amino acid residue SEQ ID NO:19; and at least two of the following: (i) an amino acid other than lysine at position 139 corresponding to amino acid residue SEQ ID NO:19, (ii) an amino acid other than glycine at position 146 corresponding to amino acid residue SEQ ID NO:19, (iii) an amino acid other than threonine at position 163 corresponding to amino acid residue SEQ ID NO:19, and (iv) an amino acid other than valine at position 164 corresponding to amino acid residue SEQ ID NO:19.

[0267] In some respects, the DRβ chain comprises (a) an amino acid other than leucine at position 114 corresponding to amino acid residue SEQ ID NO:19, (b) an amino acid other than valine at position 143 corresponding to amino acid residue SEQ ID NO:19, (c) an amino acid other than serine at position 118 corresponding to amino acid residue SEQ ID NO:19, and (d) an amino acid other than threonine at position 157 corresponding to amino acid residue SEQ ID NO:19; and at least three of the following: (i) an amino acid other than lysine at position 139 corresponding to amino acid residue SEQ ID NO:19, (ii) an amino acid other than glycine at position 146 corresponding to amino acid residue SEQ ID NO:19, (iii) an amino acid other than threonine at position 163 corresponding to amino acid residue SEQ ID NO:19, and (iv) an amino acid other than valine at position 164 corresponding to amino acid residue SEQ ID NO:19.

[0268] In some respects, the DRβ chain comprises (a) an amino acid other than leucine at position 114 corresponding to amino acid residue SEQ ID NO:19, (b) an amino acid other than valine at position 143 corresponding to amino acid residue SEQ ID NO:19, (c) an amino acid other than serine at position 118 corresponding to amino acid residue SEQ ID NO:19, (d) an amino acid other than lysine at position 139 corresponding to amino acid residue SEQ ID NO:19, (e) an amino acid other than glycine at position 146 corresponding to amino acid residue SEQ ID NO:19, (f) an amino acid other than threonine at position 157 corresponding to amino acid residue SEQ ID NO:19, (g) an amino acid other than threonine at position 163 corresponding to amino acid residue SEQ ID NO:19, and (h) an amino acid other than valine at position 164 corresponding to amino acid residue SEQ ID NO:19.

[0269] In some respects, the DRβ chain contains (a) tryptophan at position 114 of amino acid residue SEQ ID NO:19, (b) methionine at position 143 of amino acid residue SEQ ID NO:19, (c) histidine at position 118 of amino acid residue SEQ ID NO:19, and (d) isoleucine at position 157 of amino acid residue SEQ ID NO:19.

[0270] In some respects, (i) the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19, (ii) the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19, or each of the amino acid other than leucine at position 114 of amino acid residue SEQ ID NO:19 and the amino acid other than valine at position 143 of amino acid residue SEQ ID NO:19 is an amino acid containing a hydrophobic side chain.

[0271] In some respects, (i) the amino acid at position 114 corresponding to amino acid residue SEQ ID NO:19, excluding leucine, is selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan; (ii) the amino acid at position 143 corresponding to amino acid residue SEQ ID NO:19, excluding valine, is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; (iii) the amino acid at position 118 corresponding to amino acid residue SEQ ID NO:19, excluding serine, is selected from arginine, histidine, and lysine; and / or (iv) the amino acid at position 157 corresponding to amino acid residue SEQ ID NO:19, excluding threonine, is selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0272] In some respects, (i) the amino acid at position 114 of SEQ ID NO:19, excluding leucine, is selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan; (ii) the amino acid at position 143 of SEQ ID NO:19, excluding valine, is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; (iii) the amino acid at position 118 of SEQ ID NO:19, excluding serine, is selected from arginine, histidine, and lysine; (iv) the amino acid at position 157 of SEQ ID NO:19, excluding threonine, is selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; (v) the amino acid at position 139 of SEQ ID NO:19, excluding lysine, is selected from serine, threonine, and glutamine; (vi) the amino acid at position 143 of SEQ ID NO:19, excluding leucine, is selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; The amino acid at position 146 of SEQ ID NO:19, excluding glycine, is selected from serine, asparagine, threonine, and glutamine; (vii) the amino acid at position 163 of SEQ ID NO:19, excluding threonine, is selected from alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; and / or (viii) the amino acid at position 164 of SEQ ID NO:19, excluding valine, is selected from serine, asparagine, threonine, and glutamine.

[0273] In some respects, the DRβ chain described herein exhibits increased affinity for CD4 protein compared to a reference HLA class II molecule. In some respects, the reference HLA class II molecule is an HLA class II molecule having a wild-type DRβ chain. In some respects, the reference HLA class II molecule is an HLA class II molecule having a DRβ chain comprising (i) leucine at position 114 corresponding to amino acid residue SEQ ID NO:19 and / or (ii) valine at position 143 corresponding to amino acid residue SEQ ID NO:19. In some respects, the reference HLA class II molecule is an HLA class II molecule having a DRβ chain comprising (i) leucine at position 114 corresponding to amino acid residue SEQ ID NO:19, (ii) valine at position 143 corresponding to amino acid residue SEQ ID NO:19, (iii) serine at position 118 corresponding to amino acid residue SEQ ID NO:19, and (iv) threonine at position 157 corresponding to amino acid residue SEQ ID NO:19.

[0274] In some respects, the increased affinity for CD4 is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 1000 times, at least about 1500 times, at least about 2000 times, at least about 2500 times, at least about 3000 times, at least about 3500 times, at least about 4000 times, at least about 4500 times, or at least about 4000 times.

[0275] In some respects, the increased affinity for CD4 is at least approximately 1.5 to at least approximately 5000 times, 1.5 to at least approximately 4000 times, 1.5 to at least approximately 3000 times, 1.5 to at least approximately 2000 times, 1.5 to at least approximately 1000 times, 10 to at least approximately 5000 times, 10 to at least approximately 4000 times, 10 to at least approximately 3000 times, 10 to at least approximately 2000 times, 10 to at least approximately 1000 times, 10 to at least approximately 900 times, 10 to at least approximately 800 times, 10 to at least approximately 700 times, 10 to at least approximately 600 times, 10 to at least approximately 500 times, and 10 to at least approximately 500 times, respectively. At least 400 times, 10 times to at least 300 times, 10 times to at least 200 times, 10 times to at least 100 times, 100 times to at least 5000 times, 100 times to at least 4000 times, 100 times to at least 3000 times, 100 times to at least 2000 times, 100 times to at least 1000 times, 100 times to at least 900 times, 100 times to at least 800 times, 100 times to at least 700 times, 100 times to at least 600 times, 100 times to at least 500 times, 100 times to at least 400 times, 100 times to at least 300 times, or 100 times to at least 200 times.

[0276] In some respects, the DRβ chain contains alleles selected from the following: HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*06, HLA-DRB1*07, HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*10, HLA-DRB1*11, HLA-DRB1*12, HLA-DRB1*13, HLA-DRB1*14, HLA-DRB1*15, or HLA-DRB1*16. In some respects, the DRβ chain contains the HLA-DRB1*01 allele. In certain respects, the DRβ chain contains the HLA-DRB1*01:01 allele.

[0277] In some aspects, DRβ chain contains the following genes: DRB1*01:01:01、DRB1*01:01:02、DRB1*01:01:03、DRB1*01:01:04、DRB1*01:01:05、DRB1*01:01:06、DRB1*01:01:07、DRB1 *01:01:08、DRB1*01:01:09、DRB1*01:01:10、DRB1*01:01:11、DRB1*01:01:12、DRB1*01:01:13、DRB1*01:01:14、DRB1*01:01:15、DRB1*01:01:16、DRB1*01:01:16 B1*01:01:17、DRB1*01:01:18、DRB1*01:01:19、DRB1*01:01:20、DRB1*01:01:21、DRB1*01:01:22、DRB1*01:01:23、DRB1*01:01:24、DRB1*01:01:25、 DRB1*01:01:26、DRB1*01:01:27、DRB1*01:01:28、DRB1*01:01:29、DRB1*01:01:30、DRB1*01:01:31、DRB1*01:01:32、DRB1*01:01:33、DRB1*01:02:0 1:01、DRB1*01:02:01:02、DRB1*01:02:02、DRB1*01:02:03、DRB1*01:02:04、DRB1*01:02:05、DRB1*01:02:06、DRB1*01:02:07、DRB1*01:02:08、DRB1*01:02:09、DRB1*01:02:10、DRB1*01:02:11、DRB1*01:02:12、DRB1*01:02:13、DRB1*01:03:01、DRB1*01:03:02、DRB1*01:03:03、DRB1*01:03:04、DRB1*01:036、DRB1*01:03:07、DRB1*01:03:08、DRB1*01:03:09、DRB1*01:02:10、DRB1*01:02:11、DRB1*01:02:12、DRB1*01:02:13、DRB1*01:0 B1*01:04、DRB1*01:05、DRB1*01:06、DRB1*01:07、DRB1*01:08、DRB1*01:09、DRB1*01:10、DRB1*01:100、DRB1*01:11:01、DRB1*01:11:02、DRB1*01:10 2、DRB1*01:13、DRB1*01:14、DRB1*01:15、DRB1*01:16、DRB1*01:17、DRB1*01:18:01、DRB1*01:18:02、DRB1*01:19、DRB1*01:20:01、DRB1*01:20:02、DRB1*01:21、DRB1*01:22、DRB1*01:23、DRB1*01:24:01、DRB1*01:24:02、DRB1*01:25、DRB1*01:26、DRB1*01:27、DRB1*01:28、DRB1*01:29:01、DRB1*01:29:02、DRB1*01:30、DRB1*01:31、DRB1*01:32、DRB1*01:33N、DRB1*01:34、DRB1*01:35、DRB1*01:36、DRB1*01:37、DRB1*01:38、DRB1*01:39N、DRB 1*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、DR B1*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、D RB1*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*01:91、DRB1*01:92、DRB1*01:92 1*01:93、DRB1*01:94、DRB1*01:95、DRB1*01:96、DRB1*01:97、DRB1*01:98 、DRB1*01:99、DRB1*03:01:01:01、DRB1*03:01:01:02、DRB1*03:01:01:03、DRB1*03:01:02、DRB1*03:01:03、DRB1*03:01:04、DRB1*03:01:05、DRB1*03:01:06、DRB1*03:01:07、DRB1*03:01:08、DRB1*03:01:09、DRB1*03:01:10、DRB1*03:01:11、DRB1*03:01:12、DRB1*03:01:13、DRB1*03:01:14、DRB1*03:01:15、DRB1*03:01:16、DRB1*03:01:17、DRB1*03:01:18、DRB1*03:01:1 9、DRB1*03:01:20、DRB1*03:01:21、DRB1*03:01:22、DRB1*03:01:23、DRB1*03:01:24、DRB1*03:01:25、DRB1*03:01:26、DRB1*03:01:27、DRB1*03:01:28、DRB1*03:02:01、DRB1*03:02:02、DRB1*03:02:03、DRB1*03:03、DRB1*03:04:01、DRB1*03:04:02、DRB1*03:05:01、DRB1*03:05:02、DRB1*03:05:03 、DRB1*03:06、DRB1*03:07:01、DRB1*03:07:02、DRB1*03:08、DRB1*03:09、DRB1*03:10、DRB1*03:100:01、DRB1*03:100:02、DRB1*03:10、DRB1*03:10 2、DRB1*03:103、DRB1*03:104、DRB1*03:105、DRB1*03:106、DRB1*03:107、DRB1*03:108、DRB1*03:109、DRB1*03:110、DRB1*03:111、DRB1*03:112、DRB1*03:112 1*03:113、DRB1*03:114、DRB1*03:115、DRB1*03:116、DRB1*03:117、DRB1*03:118、DRB1*03:119、DRB1*03:11:01、DRB1*03:12、DRB1*03:120、DRB1*03:121、DRB1*03:122、DRB1*03:123、DRB1*03:124、DRB1*03:125、DRB1*03:126、DRB1*03:127、DRB1*03:128、DRB1*03:129、DRB1*03:130、DRB1*03:131、DRB1*03:132、DRB1*03:133、DRB1*03:134、DRB1*03:135、DRB1*03:136、DRB1*03:137、DRB1*03:138、DRB1*03:139、DRB1*03:13:01、DRB1*03:13:02、DRB1*03:14、DRB1*03:140、DRB1*03:141、DRB1*03:142、DRB1*03:143、DRB1*03:144、DRB1*03:145、DRB1*03:146、DRB1*03:147、DRB1*03:148、DRB1*0 3:149、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*03:73 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、D RB1*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*04:01:03、DRB1*04:01:02、DRB1*04:01:03、DRB1*04:01:04、DRB1*04:01:05、DRB1*04:01:06、DRB1*04:0 1:07、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*04:0 5:01:02、DRB1*04:05:01:03、DRB1*04:05:02、DRB1*04:05:03、DRB1*04:0 5:04、DRB1*04:05:05、DRB1*04:05:06、DRB1*04:05:07、DRB1*04:05:08、DR B1*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、D RB1*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*04:1 25、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*04:19 8、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 1、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、D RB1*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*04:262 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*04:269 272、DRB1*04:28、DRB1*04:29、DRB1*04:30、DRB1*04:31、DRB1*04:32、DRB1*04:33、DRB1*04:34、DRB1*04:35、DRB1*04:36、DRB1*04:37、DRB1*04:38、DRB1*04:39、DRB1*04:40、DRB1*04:41、DRB1*04:42、DRB1*04:43、DRB1*04:44:01、DRB1*04:44:02、DRB1*04:45、DRB1*04:46、DRB1*04:47、DRB1*04:4 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1*11:219、DRB1*11:22、DRB1*11:220、DRB1*11:221、DRB1*11:222、DRB1*11:223、DRB1*11:224、DRB1*11:225、DRB1*11:226、DRB1*11:227、DRB1*11:224 228、DRB1*11:229、DRB1*11:230、DRB1*11:231、DRB1*11:232、DRB1*11:233、DRB1*11:234、DRB1*11:235、DRB1*11:236、DRB1*11:237、DRB1*11:238、DRB1*11:239、DRB1*11:23:01、DRB1*11:23:02、DRB1*11:240、DRB1*11:241、DRB1*11:242、DRB1*11:243、DRB1*11:244、DRB1*11:245、DRB1*11:246N 、DRB1*11:247、DRB1*11:248Q、DRB1*11:249、DRB1*11:24:01、DRB1*11:24:02、DRB1*11:25、DRB1*11:250N、DRB1*11:251、DRB1*11:252、DRB1*11:25 3、DRB1*11:254、DRB1*11:26、DRB1*11:27:01、DRB1*11:27:02、DRB1*11:27:03、DRB1*11:28:01、DRB1*11:28:02、DRB1*11:29:01、DRB1*11:29:02、D RB1*11:30、DRB1*11:31、DRB1*11:32、DRB1*11:33、DRB1*11:34、DRB1*11:35、DRB1*11:36、DRB1*11:37:01、DRB1*11:37:02、DRB1*11:38、DRB1*11:39、DRB1*11:40、DRB1*11:41、DRB1*11:42:01、DRB1*11:42:02、DRB1*11:43、DRB1*11:44、DRB1*11:45、DRB1*11:46:01、DRB1*11:46:02、DRB1*11:47、DRB1*11:48、DRB1*11:49:01、DRB1*11:49:02、DRB1*11:50、DRB1*11:51、DRB1*11:52、DRB1*11:53、DRB1*11:54:01、DRB1*11:54:02、DRB1*11:55、DRB1*11:55 D RB1*11:63:02、DRB1*11:64、DRB1*11:65:01、DRB1*11:65:02、DRB1*11:66:01、DRB1*11:66:02、DRB1*11:67、DRB1*11:68、DRB1*11:69、DRB1*11:70、DRB1*11:72、DRB1*11:73、DRB1*11:74:01、DRB1*11:74:02、DRB1*11:75、DRB1*11:76、DRB1*11:77、DRB1*11:78、DRB1*11:79、DRB1*11:80、DRB1*11: 81、DRB1*11:82、DRB1*11:83、DRB1*11:84:01、DRB1*11:84:02、DRB1*11:84:03、DRB1*11:85、DRB1*11:86、DRB1*11:87、DRB1*11:88、DRB1*11:89、DRB1*11:90、DRB1*11:91、DRB1*11:92、DRB1*11:93、DRB1*11:94、DRB1*11:95、DRB1*11:96、DRB1*11:97、DRB1*11:98、DRB1*11:99、DRB1*12:01:01:01 ,DRB1*12:01:01:02,DRB1*12:01:01:03,DRB1*12:01:01:04,DRB1*12:01:01:05,DRB1*12:01:01:06,DRB1*12:01:02,DRB1*12:01:03,DRB1*12:01:04,DRB1*12:01:05,DRB1*12:01:06,DRB1*12:01:07,DRB1*12:01:08,DRB1*12:01:09,DRB1*12:02:01:01,DRB1*12:02:01:02,DRB1*12:02:01:03,DRB1*12:02:01:04、DRB1*12:02:02、DRB1*12:02:03、DRB1*12:02:04、DRB1*12:02:05、DRB1*12:02:06、DRB1*12:02:07、DRB1*12:02:08、DRB1*12:08 02:09、DRB1*12:03:02、DRB1*12:03:03、DRB1*12:04、DRB1*12:05、DRB1*12:06、DRB1*12:07、DRB1*12:08、DRB1*12:09、DRB1*12:10、DRB1*12:11、DRB1*12:11 RB1*12:12、DRB1*12:13、DRB1*12:14、DRB1*12:15、DRB1*12:16:01、DRB1*12:16:02、DRB1*12:16:03、DRB1*12:17、DRB1*12:18、DRB1*12:19、DRB1*12:20、DRB1*12:21、DRB1*12:22、DRB1*12:23、DRB1*12:24N、DRB1*12:25、DRB1*12:26、DRB1*12:27、DRB1*12:28、DRB1*12:29、DRB1*12:30、DRB1*1 2:31N, DRB1*12:32, DRB1*12:33, DRB1*12:34, DRB1*12:35, DRB1*12:36, DRB1*12:37, DRB1*12:38, DRB1*12:39, DRB1*12:40, DRB1*12:41, DRB1*12:42, DRB1*12:43, DRB1*12:44, DRB1*12:45, DRB1*12:46, DRB1*12:47, DRB1*12:48, DRB1*12:49, DRB1*12:50, DRB1*12:51, DRB1*12:52, DRB1*12:5 3、DRB1*12:54、DRB1*12:55、DRB1*12:56、DRB1*12:57、DRB1*12:58、DRB1*12:59、DRB1*12:60N、DRB1*12:61、DRB1*12:62、DRB1*12:63、DRB1*12:64 、DRB1*12:65、DRB1*12:66、DRB1*12:67、DRB1*12:68、DRB1*12:69、DRB1*12:70、DRB1*12:71、DRB1*12:72N、DRB1*12:73、DRB1*12:74N、DRB1*12:75、DRB1*13:01:01:01、DRB1*13:01:01:02、DRB1*13:01:02、DRB1*13:01:03、DRB1*13:01:04、DRB1*13:01:05、DRB1*13:01:06、DRB1*13:01:07、DRB1*13:01:08、DRB1*13:01:09、DRB1*13:01:10、DRB1*13:01:11、DRB1*13:01:12、DRB1*13:01:13、DRB1*13:01:14、DRB1*13:01:15、DRB1*13:01:16、DRB1 *13:01:17、DRB1*13:01:18、DRB1*13:01:19、DRB1*13:01:20、DRB1*13:01:21、DRB1*13:01:22、DRB1*13:01:23、DRB1*13:01:24、DRB1*13:01:25、DRB1*13:01:26、DRB1*13:02:01:01、DRB1*13:02:01:02、DRB1*13:02:01:03、DRB1*13:02:02、DRB1*13:02:03、DRB1*13:02:04、DRB1*13:02:05、DRB1*1 3:02:06、DRB1*13:02:07、DRB1*13:02:08、DRB1*13:02:09、DRB1*13:02:10、DRB1*13:02:11、DRB1*13:02:12、DRB1*13:02:13、DRB1*13:02:14、DRB1 *13:02:15、DRB1*13:02:16、DRB1*13:02:17、DRB1*13:03:01、DRB1*13:03:02、DRB1*13:03:03、DRB1*13:03:04、DRB1*13:03:05、DRB1*13:03:06、DRB1*13:03:06 B1*13:03:07、DRB1*13:03:08、DRB1*13:03:09、DRB1*13:04、DRB1*13:05:01、DRB1*13:05:02、DRB1*13:05:03、DRB1*13:06、DRB1*13:07:01、DRB1*13:07:02、DRB1*13:08、DRB1*13:09、DRB1*13:10、DRB1*13:100、DRB1*13:101、DRB1*13:102、DRB1*13:103、DRB1*13:104、DRB1*13:105、DRB1*13:106、DRB1*13:107、DRB1*13:108、DRB1*13:109、DRB1*13:110、DRB1*13:111、DRB1*13:112、DRB1*13:113N、DRB1*13:114、DRB1*13:115、DRB1*13:116、DRB1*13:116 1*13:117、DRB1*13:118、DRB1*13:119、DRB1*13:11:01、DRB1*13:11:02、DRB1*13:120、DRB1*13:121、DRB1*13:122、DRB1*13:123、DRB1*13:124、DRB1*13:124 1*13:125、DRB1*13:126、DRB1*13:127、DRB1*13:128、DRB1*13:129、DRB1*13:12:01、DRB1*13:12:02、DRB1*13:12:03、DRB1*13:12:04、DRB1*13:13、DRB1*13:130、DRB1*13:131、DRB1*13:132、DRB1*13:133、DRB1*13:134、DRB1*13:135、DRB1*13:136、DRB1*13:137N、DRB1*13:138、DRB1*13:139、DRB1 *13:140、DRB1*13:141、DRB1*13:142N、DRB1*13:143、DRB1*13:144、DRB1*13:145、DRB1*13:146、DRB1*13:147、DRB1*13:148、DRB1*13:149、DRB1*13:14:01、DRB1*13:14:02、DRB1*13:14:03、DRB1*13:15、DRB1*13:150、DRB1*13:151、DRB1*13:152、DRB1*13:153、DRB1*13:154、DRB1*13:155、DRB1*13 :156、DRB1*13:157、DRB1*13:158、DRB1*13:159、DRB1*13:16、DRB1*13:160、DRB1*13:161、DRB1*13:162、DRB1*13:163、DRB1*13:164、DRB1*13:165、DRB1*13:166、DRB1*13:167、DRB1*13:168、DRB1*13:169、DRB1*13:17、DRB1*13:170、DRB1*13:171:01、DRB1*13:171:02、DRB1*13:172、DRB1*13:173、DRB1*13:174、DRB1*13:175、DRB1*13:176、DRB1*13:177、DRB1*13:178、DRB1*13:179、DRB1*13:18、DRB1*13:180、DRB1*13:181、DRB1*13:182、DRB1*1 3:183、DRB1*13:184、DRB1*13:185N、DRB1*13:186、DRB1*13:187、DRB1*13:188、DRB1*13:189、DRB1*13:19、DRB1*13:190、DRB1*13:191、DRB1*13:192 、DRB1*13:193、DRB1*13:194、DRB1*13:195、DRB1*13:196、DRB1*13:197、DRB1*13:198、DRB1*13:199、DRB1*13:20、DRB1*13:200、DRB1*13:201、DRB1*13:201 *13:202、DRB1*13:203、DRB1*13:204、DRB1*13:205、DRB1*13:206、DRB1*13:207、DRB1*13:208、DRB1*13:209、DRB1*13:210、DRB1*13:211、DRB1*13:2 12、DRB1*13:213、DRB1*13:214、DRB1*13:215、DRB1*13:216、DRB1*13:217、DRB1*13:218、DRB1*13:219、DRB1*13:21:01、DRB1*13:21、DRB1*13:22 0. RB1*13:22:02, DRB1*13:230, DRB1*13:231, DRB1*13:232, DRB1*13:233, DRB1*13:234, DRB1*13:235, DRB1*13:236, DRB1*13:237, DRB1*13:238, DRB1*13:239, DRB1*13:23:01, DRB1*13:23:02, DRB1*13:24, DRB1*13:240, DRB1*13:241, DRB1*13:242:01, DRB1*13:242:02, DRB1*13:243, DRB1*13:244,DRB1*13:245、DRB1*13:246、DRB1*13:247、DRB1*13:248、DRB1*13:249N、DRB1*13:25、DRB1*13:250、DRB1*13:251、DRB1*13:252N、DRB1*13:253、DRB1*13:254、DRB1*13:255N、DRB1*13:256、DRB1*13:257、DRB1*13:258、DRB1*13:259、DRB1*13:260、DRB1*13:261、DRB1*13:262、DRB1*13:263、DRB1* 13:264、DRB1*13:265、DRB1*13:266、DRB1*13:267、DRB1*13:268N、DRB1*13:269、DRB1*13:26:01、DRB1*13:26:02、DRB1*13:27、DRB1*13:270、DRB1 *13:271、DRB1*13:272、DRB1*13:273、DRB1*13:274、DRB1*13:275、DRB1*13:276、DRB1*13:277、DRB1*13:278Q、DRB1*13:279、DRB1*13:280、DRB1*13:278 13:28:02, DRB1*13:29, DRB1*13:30, DRB1*13:31, DRB1*13:32, DRB1*13:33:01, DRB1*13:33:02, DRB1*13:33:03, DRB1*13:34, DRB1*13:35, DRB1*13:36, DRB1*13:37, DRB1*13:38, DRB1*13:39, DRB1*13:40, DRB1*13:41, DRB1*13:42, DRB1*13:43, DRB1*13:44, DRB1*13:45, DRB1*13:46, DRB1*13:4 7、DRB1*13:48、DRB1*13:49、DRB1*13:50:01、DRB1*13:50:02、DRB1*13:50:03、DRB1*13:51、DRB1*13:52、DRB1*13:53、DRB1*13:54、DRB1*13:55、DRB1*13:56、DRB1*13:57、DRB1*13:58、DRB1*13:59、DRB1*13:60、DRB1*13:61:01、DRB1*13:61:02、DRB1*13:62、DRB1*13:63、DRB1*13:64、DRB1*13:65、DRB1*13:66:01、DRB1*13:66:02、DRB1*13:67、DRB1*13:68、DRB1*13:69、DRB1*13:70、DRB1*13:71、DRB1*13:72、DRB1*13:73、DRB1*13:74、DRB1*13:71 75、DRB1*13:76、DRB1*13:77、DRB1*13:78、DRB1*13:79、DRB1*13:80、DRB1*13:81、DRB1*13:82、DRB1*13:83、DRB1*13:84、DRB1*13:85、DRB1*13:86、D RB1*13:87、DRB1*13:88、DRB1*13:89:01、DRB1*13:89:02、DRB1*13:90、DRB1*13:91、DRB1*13:92、DRB1*13:93、DRB1*13:94:01、DRB1*13:94:02、DRB1*13:95、DRB1*13:96:01、DRB1*13:96:02、DRB1*13:97:01、DRB1*13:97:02、DRB1*13:98、DRB1*13:99、DRB1*14:01:01、DRB1*14:01:02、DRB1*14:01:0 3、DRB1*14:01:04、DRB1*14:02:01:01、DRB1*14:02:01:02、DRB1*14:02:02、DRB1*14:02:03、DRB1*14:02:04、DRB1*14:02:05、DRB1*14:02:06、DRB1 *14:02:07、DRB1*14:03:01、DRB1*14:03:02、DRB1*14:04:01、DRB1*14:04:02、DRB1*14:04:03、DRB1*14:04:04、DRB1*14:04:05、DRB1*14:04:06、DRB1*14:04:06 1*14:05:01:01, DRB1*14:05:01:02, DRB1*14:05:02, DRB1*14:05:03, DRB1*14:05:04, DRB1*14:06:01, DRB1*14:06:02, DRB1*14:06:03, DRB1*14:06:04, DRB1*14:07:01, DRB1*14:07:02, DRB1*14:08, DRB1*14:09, DRB1*14:10, DRB1*14:100, DRB1*14:101, DRB1*14:102, DRB1*14:103, DRB1*14:104,DRB1*14:105、DRB1*14:106、DRB1*14:107、DRB1*14:108、DRB1*14:109、DRB1*14:11、DRB1*14:110、DRB1*14:111、DRB1*14:112、DRB1*14:113、DRB1*14:114、DRB1*14:115、DRB1*14:116、DRB1*14:117、DRB1*14:118、DRB1*14:119、DRB1*14:120、DRB1*14:121、DRB1*14:122、DRB1*14:123、DRB1*14:12 4、DRB1*14:125、DRB1*14:126:01、DRB1*14:126:02、DRB1*14:127:01、DRB1*14:127:02、DRB1*14:128、DRB1*14:129、DRB1*14:12:01、DRB1*14:12:02 2、DRB1*14:13、DRB1*14:130、DRB1*14:131、DRB1*14:132、DRB1*14:133、DRB1*14:134、DRB1*14:135、DRB1*14:136、DRB1*14:137、DRB1*14:138、DRB1*14:138 1*14:139、DRB1*14:14、DRB1*14:140、DRB1*14:141、DRB1*14:142、DRB1*14:143、DRB1*14:144、DRB1*14:145、DRB1*14:146、DRB1*14:147、DRB1*14:144 148、DRB1*14:149、DRB1*14:15、DRB1*14:150、DRB1*14:151、DRB1*14:152N、DRB1*14:153、DRB1*14:154、DRB1*14:155、DRB1*14:156、DRB1*14:157、D RB1*14:158、DRB1*14:159、DRB1*14:16、DRB1*14:160、DRB1*14:161、DRB1*14:162、DRB1*14:163、DRB1*14:164、DRB1*14:165、DRB1*14:166N、DRB1*14:167、DRB1*14:168、DRB1*14:169、DRB1*14:17、DRB1*14:170、DRB1*14:171、DRB1*14:172、DRB1*14:173、DRB1*14:174、DRB1*14:175、DRB1*14:176、DRB1*14:177、DRB1*14:178、DRB1*14:179、DRB1*14:18、DRB1*14:180、DRB1*14:181、DRB1*14:182、DRB1*14:183、DRB1*14:184、DRB1*14:185、DRB1*14:186、DRB1*14:187、DRB1*14:188N、DRB1*14:189、DRB1*14:19、DRB1*14:190、DRB1*14:191、DRB1*14:192、DRB1*14:193、DRB1*14:194、DRB1*14:1 95N、DRB1*14:196、DRB1*14:197N、DRB1*14:198、DRB1*14:199、DRB1*14:20、DRB1*14:200、DRB1*14:201、DRB1*14:202、DRB1*14:203、DRB1*14:204、DRB1*14:205、DRB1*14:206、DRB1*14:207、DRB1*14:208、DRB1*14:209、DRB1*14:21、DRB1*14:210Q、DRB1*14:211、DRB1*14:22、DRB1*14:23:01、DRB1 *14:23:02、DRB1*14:23:03、DRB1*14:23:04、DRB1*14:24、DRB1*14:25:01、DRB1*14:25:02、DRB1*14:26、DRB1*14:27:01、DRB1*14:27:02、DRB1*14:26 28、DRB1*14:29、DRB1*14:30、DRB1*14:31、DRB1*14:32:01、DRB1*14:32:02、DRB1*14:32:03、DRB1*14:33、DRB1*14:34、DRB1*14:35、DRB1*14:36、DRB1*14:36 1*14:37, DRB1*14:38:01, DRB1*14:38:02, DRB1*14:39, DRB1*14:40, DRB1*14:41, DRB1*14:42, DRB1*14:43, DRB1*14:44:01, DRB1*14:44:02, DRB1*14:44:03, DRB1*14:45, DRB1*14:46, DRB1*14:47, DRB1*14:48, DRB1*14:49, DRB1*14:50, DRB1*14:51, DRB1*14:52, DRB1*14:53, DRB1*14:54:01:01,DRB1*14:54:01:02、DRB1*14:54:01:03、DRB1*14:54:01:04、DRB1*14:54:02、DRB1*14:54:03、DRB1*14:54:04、DRB1*14:54:05、DRB1*14:54:06、DRB1*14:54:06 B1*14:54:07、DRB1*14:55、DRB1*14:56、DRB1*14:57、DRB1*14:58、DRB1*14:59、DRB1*14:60、DRB1*14:61、DRB1*14:62、DRB1*14:63、DRB1*14:64、DRB1*14:64 B1*14:65、DRB1*14:67、DRB1*14:68:01、DRB1*14:68:02、DRB1*14:69、DRB1*14:70、DRB1*14:71、DRB1*14:72、DRB1*14:73、DRB1*14:74、DRB1*14:7 5、DRB1*14:76、DRB1*14:77、DRB1*14:78、DRB1*14:79、DRB1*14:80、DRB1*14:81、DRB1*14:82、DRB1*14:83、DRB1*14:84、DRB1*14:85、DRB1*14:86、D RB1*14:87、DRB1*14:88、DRB1*14:89、DRB1*14:90、DRB1*14:91、DRB1*14:92N、DRB1*14:93、DRB1*14:94、DRB1*14:95、DRB1*14:96、DRB1*14:97、DRB1*14:98、DRB1*14:99、DRB1*15:01:01:01、DRB1*15:01:01:02、DRB1*15:01:01:03、DRB1*15:01:01:04、DRB1*15:01:01:05、DRB1*15:01:02、DRB1* 15:01:03, DRB1*15:01:04, DRB1*15:01:05, DRB1*15:01:06, DRB1*15:01:07, DRB1*15:01:08, DRB1*15:01:09, DRB1*15:01:10, DRB1*15:01:11, DRB1*15:01:12, DRB1*15:01:13, DRB1*15:01:14, DRB1*15:01:15, DRB1*15:01:16, DRB1*15:01:17, DRB1*15:01:18, DRB1*15:01:19, DRB1*15:01:20,DRB1*15:01:21、DRB1*15:01:22、DRB1*15:01:23、DRB1*15:01:24、DRB1*15:01:25、DRB1*15:01:26、DRB1*15:01:27、DRB1*15:01:28、DRB1*15:01:29、DRB1*15:01:30、DRB1*15:01:31、DRB1*15:01:32、DRB1*15:01:33、DRB1*15:01:34、DRB1*15:01:35、DRB1*15:01:36、DRB1*15:01:37、DRB1*15:0 1:38、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、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*15:06:02 B1*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*15:117 B1*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、DRB 1*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*15:1 43、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*1 5:46、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*15:52 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、DRB1*15:02、DRB1*15:02 5:67、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*15:7 8、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、D RB1*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*16:07 2:08、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*1 6:09:02、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*16:27 1*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.,

[0278] In some respects, MHC class II molecules comprise a DRβ chain containing 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 with SEQ ID NO:21, wherein the DRβ chain comprises (i) tryptophan at position 114 corresponding to amino acid residue 114 of SEQ ID NO:19, (ii) methionine at position 143 corresponding to amino acid residue 143 of SEQ ID NO:19, (iii) histidine at position 118 corresponding to amino acid residue 118 of SEQ ID NO:19, and (vi) isoleucine at position 157 corresponding to amino acid residue 157 of SEQ ID NO:19. In some respects, MHC class II molecules comprise a DRβ chain containing 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 with SEQ ID NO:21, wherein the DRβ chain comprises (i) tryptophan at position 114 corresponding to amino acid residue 114 of SEQ ID NO:19, (ii) methionine at position 143 corresponding to amino acid residue 114 of SEQ ID NO:19, (iii) histidine at position 118 corresponding to amino acid residue 118 of SEQ ID NO:19, (iv) isoleucine at position 157 corresponding to amino acid residue 119, (v) threonine at position 139 corresponding to amino acid residue 119, (vi) glutamine at position 146 corresponding to amino acid residue 119, and (vii) other amino acids at position 114 corresponding to amino acid residue 118 of SEQ ID NO:19. Methionine at position 163 of amino acid residue NO:19; and (vii) threonine at position 164 of amino acid residue corresponding to SEQ ID NO:19. In some respects, MHC class II molecules contain a DRβ chain containing the amino acid sequence shown in SEQ ID NO:21.

[0279] II.A.3.b. HLA-DRα chain

[0280] In some aspects of this disclosure, MHC class II molecules also comprise an α chain. In some aspects, the α chain is a wild-type α chain. In some aspects, the α chain is a DRα chain. Any DRα chain may be used in the compositions and methods of this disclosure. In some aspects, the DRα chain comprises the HLA-DRA1*01 allele.

[0281] In some respects, the DRα 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.

[0282] In some aspects, MHC class II molecules comprise a DRα chain containing 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 with SEQ ID NO:24. In some aspects, MHC class II molecules comprise a DRα chain containing 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 with SEQ ID NO:26. In some aspects, MHC class II molecules comprise a DRα chain containing the amino acid sequence shown in SEQ ID NO:24. In some respects, MHC class II molecules contain a DRα chain, which contains the amino acid sequence shown in SEQ ID NO:26.

[0283] II.A.4 Signal Peptide

[0284] In some aspects, the β chain and / or α chain further comprises a signal peptide. Any signal peptide known in the art may be used in the compositions and methods disclosed herein. In some aspects, the β chain signal peptide is identical to the α signal peptide. In some aspects, the β chain signal peptide differs from the α signal peptide.

[0285] In some respects, signal peptides are derived from naturally occurring signal peptides. In some respects, signal peptides are derived from naturally occurring DPβ chain signal peptides. In some respects, signal peptides contain naturally occurring DPβ chain signal peptides. In some respects, signal peptides are derived from naturally occurring DPα chain signal peptides. In some respects, signal peptides contain naturally occurring DPα chain signal peptides.

[0286] In some respects, the signal peptide is derived from a naturally occurring DQβ chain signal peptide. In some respects, the signal peptide comprises a naturally occurring DQβ chain signal peptide. In some respects, the signal peptide is derived from a naturally occurring DQα chain signal peptide. In some respects, the signal peptide comprises a naturally occurring DQα chain signal peptide.

[0287] In some respects, signal peptides are derived from naturally occurring DRβ chain signal peptides. In some respects, signal peptides contain naturally occurring DRβ chain signal peptides. In some respects, signal peptides are derived from naturally occurring DRα chain signal peptides. In some respects, signal peptides contain naturally occurring DRα chain signal peptides.

[0288] In some respects, the signal peptide is derived from the fibrin light chain (FibL) signal peptide. In some respects, the signal peptide comprises SEQ ID NO:9. In some respects, the signal peptide is synthetic.

[0289] II.A.5. Transmembrane domain

[0290] In some aspects, the β chain and / or α chain further comprises a transmembrane domain. The transmembrane domain can be of any length and of any origin. In some aspects, the transmembrane domain is at least about 1 to at least about 50 amino acids long. 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.

[0291] In some respects, the transmembrane domains originate from naturally occurring DPβ chain transmembrane domains. In some respects, the transmembrane domains contain naturally occurring DPβ chain transmembrane domains. In some respects, the transmembrane domains originate from naturally occurring DPα chain transmembrane domains. In some respects, the transmembrane domains contain naturally occurring DPα chain transmembrane domains.

[0292] In some respects, the transmembrane domain originates from a naturally occurring DQβ chain transmembrane domain. In some respects, the transmembrane domain comprises a naturally occurring DQβ chain transmembrane domain. In some respects, the transmembrane domain originates from a naturally occurring DQα chain transmembrane domain. In some respects, the transmembrane domain comprises a naturally occurring DQα chain transmembrane domain.

[0293] In some respects, the transmembrane domains originate from naturally occurring DRβ chain transmembrane domains. In some respects, the transmembrane domains contain naturally occurring DRβ chain transmembrane domains. In some respects, the transmembrane domains originate from naturally occurring DRα chain transmembrane domains. In some respects, the transmembrane domains contain naturally occurring DRα chain transmembrane domains.

[0294] II.A.6 Leucine Zipper

[0295] In some aspects, the β-chain and / or α-chain further comprises one or more leucine zipper (LZip) sequences. Any LZip sequence known in the art may be used in the compositions and methods disclosed herein. In some aspects, the β-chain and / or α-chain comprises an acidic LZip (αLZip), a basic LZip (βLZip), or both. In some aspects, one or more LZip sequences are derived from naturally occurring LZip sequences. In some aspects, one or more LZip sequences comprise naturally occurring LZip sequences. In some aspects, one or more LZip sequences are synthetic. In some aspects, one or more LZip sequences comprise the LZip sequence shown in SEQ ID NO: 4, 7, 14, 17, 22, or 25.

[0296] II.A.7. Connector

[0297] In some aspects, the β-chain and / or α-chain used in this disclosure may further include a linker. Any linker known in the art may be used in the compositions and methods disclosed herein. In some 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 located at the N-terminus of the extracellular domain of the α-chain or β-chain. In some aspects, the linker is located at the C-terminus of the extracellular domain of the α-chain or β-chain. In some aspects, the linker is located between the extracellular domain of the α-chain or β-chain and a transmembrane domain. In some aspects, the linker is located between the extracellular domain of the α-chain or β-chain and one or more LZip sequences. In some aspects, the linker is located between the extracellular domain of the α-chain or β-chain and a signal peptide.

[0298] Linkers of any length can be used in the compositions and methods disclosed herein. In some aspects, the length of the linker is at least one amino acid. In some respects, the length of the linker is at least about 1 to at least about 100, at least about 1 to at least about 90, at least about 1 to at least about 80, at least about 1 to at least about 70, at least about 1 to at least about 60, at least about 1 to at least about 50, at least about 1 to at least about 40, at least about 1 to at least about 30, at least about 1 to at least about 20, at least about 1 to at least about 15, at least about 1 to at least about 14, at least about 1 to at least about 13, at least about 1 to at least about 12, at least about 1 to at least about 11, at least about 1 to at least about 10, at least about 1 to at least about 9, at least about 1 to at least about 8, at least about 1 to at least about 7, at least about 1 to at least about 6, at least about 1 to at least about 5, at least about 1 to at least about 4, at least about 1 to at least about 3 amino acids.

[0299] In some aspects, the length of the linker is at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids. In some aspects, the length of the linker is about 3 amino acids. In some aspects, the length of the linker is about 4 amino acids. In some aspects, the length of the linker is about 5 amino acids.

[0300] II.B cells

[0301] In some aspects of this disclosure, the MHC class II molecules used in the methods of this disclosure are attached to or associated with the cell membrane. Therefore, some aspects of this disclosure relate to methods for identifying MHC class II specific TCRs, the methods comprising contacting T cells with cells containing a complex comprising the MHC class II molecules disclosed herein and peptides (e.g., epitopes). In some aspects, the β chain of the MHC class II molecule is attached to or associated with the cell membrane. In some aspects, the α chain of the MHC class II molecule is attached to or associated with the cell membrane. In some aspects, both the α and β chains of the MHC class II molecule are attached to or associated with the cell membrane.

[0302] Any cell may be used in the methods described herein. In some respects, the cell is a mammalian cell. In some respects, the cell is an insect cell. In some respects, the cell is derived from a healthy cell, such as a healthy fibroblast. In some respects, the cell is derived from a tumor cell. Non-limiting examples of cells that may be used in this disclosure include K562 cells, T2 cells, HEK293 cells, HEK293T cells, A375 cells, SK-MEL-28 cells, Me275 cells, COS cells, fibroblasts, tumor cells, or any combination thereof. In some respects, the cell is any cell disclosed in Hasan et al., Adv. Genet. Eng. 4(3):130 (2015) (incorporated herein by reference in its entirety).

[0303] In some respects, the cell is a professional APC. In other respects, the cell is a macrophage, B cell, dendritic cell, or any combination thereof.

[0304] In some respects, the cells lack endogenous expression of one or more MHC class II alleles. In some respects, the cells lack endogenous expression of the HLA-DP allele. In some respects, the cells lack endogenous expression of the HLA-DPα chain allele. In some respects, the cells lack endogenous expression of the HLA-DPβ chain allele.

[0305] II.C. Soluble MHC Class II molecules

[0306] In some respects, the MHC class II molecules used in the methods disclosed herein do not associate with cell membranes; for example, MHC class II molecules are in a soluble form. As used herein, soluble MHC class II molecules include any MHC class II molecules or portions thereof that do not associate with cell membranes as described herein. In some respects, MHC class II molecules or portions thereof do not bind to any membrane. In some respects, MHC class II molecules or portions thereof bind to inert particles. In some respects, MHC class II molecules or portions thereof bind to the membrane of extracellular vesicles. In some respects, MHC class II molecules bind to artificial membranes or artificial surfaces, such as the surface of an array plate.

[0307] Any inert particles known in the art may be used in the compositions and methods disclosed herein. In some aspects, the inert particles are beads. In some aspects, the beads are glass beads, latex beads, metal beads, or any combination thereof. In some aspects, the inert particles are nanoparticles (NPs). Any NPs known in the art may be used in the compositions and methods disclosed herein. In some aspects, the nanoparticles are selected from polyethylene glycolated iron oxide, chitosan, dextran, gelatin, alginate, liposomes, starch, branched polymers, carbon-based carriers, polylactic acid, poly(cyano)acrylate, polyethyleneimine, block copolymers, polycaprolactone, SPIONs, USPIONs, Cd / Zn-selenide, or silica nanoparticles. In a particular aspect, the nanoparticles are polyethylene glycolated iron oxide nanoparticles. Non-limiting examples of nanoparticles that may be used in the compositions and methods disclosed herein include those set forth in De Jong and Borm, Int. J. Nanomedicine 3(2):133-49 (2008) and Umeshappa et al., Nat. Commun. 10(1):2150 (May 14, 2019) (each incorporated herein by reference in its entirety).

[0308] In some aspects, MHC class II molecules comprise fragments of full-length MHC class II molecules, wherein one or more amino acids of the transmembrane domain of the α chain and / or the transmembrane domain of the β chain are missing. In some aspects, MHC class II molecules contain an extracellular domain of the α chain (e.g., as shown in SEQ ID NO:6) and / or an extracellular domain of the β chain (e.g., as shown in SEQ ID NO:1 or 3). In some aspects, MHC class II molecules contain an extracellular domain of the α chain (e.g., as shown in SEQ ID NO:16) and / or an extracellular domain of the β chain (e.g., as shown in SEQ ID NO:11 or 13). In some aspects, MHC class II molecules contain an extracellular domain of the α chain (e.g., as shown in SEQ ID NO:24) and / or an extracellular domain of the β chain (e.g., as shown in SEQ ID NO:19 or 21).

[0309] In some aspects, MHC class II molecules comprise a DPα chain containing 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 with SEQ ID NO:6. In some aspects, MHC class II molecules comprise a DPα chain containing the amino acid sequence shown in SEQ ID NO:6.

[0310] In some aspects, MHC class II molecules comprise a DQα chain containing 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 with SEQ ID NO:16. In some aspects, MHC class II molecules comprise a DQα chain containing the amino acid sequence shown in SEQ ID NO:16.

[0311] In some aspects, MHC class II molecules comprise a DRα chain containing 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 with SEQ ID NO:24. In some aspects, MHC class II molecules comprise a DRα chain containing the amino acid sequence shown in SEQ ID NO:24.

[0312] In some aspects, MHC class II molecules comprise a DPβ chain containing 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 with SEQ ID NO:1. In some aspects, MHC class II molecules comprise a DPβ chain containing an amino acid sequence showing the amino acid sequence of SEQ ID NO:1. In some aspects, MHC class II molecules comprise a DPβ chain containing 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 with SEQ ID NO:3. In some aspects, MHC class II molecules comprise a DPβ chain containing an amino acid sequence showing the amino acid sequence of SEQ ID NO:3. In some aspects, MHC class II molecules comprise a DPβ chain containing 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 with SEQ ID NO:4. In some aspects, MHC class II molecules comprise a DPβ chain containing 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 with SEQ ID NO:5. In some aspects, MHC class II molecules comprise a DPβ chain containing an amino acid sequence shown in SEQ ID NO:5.

[0313] In some aspects, MHC class II molecules comprise a DQβ chain, said DQβ chain containing 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 with SEQ ID NO:11. In some aspects, MHC class II molecules comprise a DQβ chain, said DQβ chain containing 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 with SEQ ID NO:13. In some aspects, MHC class II molecules comprise a DQβ chain, said DQβ chain containing an amino acid sequence shown in SEQ ID NO:13. In some aspects, MHC class II molecules comprise a DQβ chain, said DQβ 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 with SEQ ID NO:14. In some aspects, MHC class II molecules comprise a DQβ chain, said DQβ 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 with SEQ ID NO:15. In some aspects, MHC class II molecules comprise a DQβ chain, said DQβ chain comprising an amino acid sequence shown in SEQ ID NO:15.

[0314] In some aspects, MHC class II molecules comprise a DRβ chain containing 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 with SEQ ID NO:19. In some aspects, MHC class II molecules comprise a DRβ chain containing an amino acid sequence showing the amino acid sequence of SEQ ID NO:19. In some aspects, MHC class II molecules comprise a DRβ chain containing 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 with SEQ ID NO:21. In some aspects, MHC class II molecules comprise a DRβ chain containing an amino acid sequence showing the amino acid sequence of SEQ ID NO:21. In some aspects, MHC class II molecules comprise a DRβ chain containing 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 with SEQ ID NO:22. In some aspects, MHC class II molecules comprise a DRβ chain containing an amino acid sequence shown in SEQ ID NO:22. In some aspects, MHC class II molecules comprise a DRβ chain containing 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 with SEQ ID NO:23. In some aspects, MHC class II molecules comprise a DRβ chain containing an amino acid sequence shown in SEQ ID NO:23.

[0315] II.D. Nucleic Acid Molecules and Vectors

[0316] Certain aspects of this disclosure relate to nucleic acid molecules encoding the MHC class II molecules disclosed herein. In some aspects, the nucleic acid molecules encode the MHC class II β chains disclosed herein. In some aspects, the nucleic acid molecules encoding the MHC class II β chains comprise nucleotide sequences 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 the sequence shown in SEQ ID NO:2, 12, or 20.

[0317] In some respects, the nucleic acid molecule encodes the MHC class II α chain disclosed herein. In some respects, the nucleic acid molecule encoding the MHC class II α 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 the sequence shown in SEQ ID NO:7, 17, or 25.

[0318] In some respects, the nucleic acid molecule encodes both the MHC class II α strand and the MHC class II β strand disclosed herein. In some respects, the sequence encoding the MHC class II α strand is under the control of the same promoter as the sequence encoding the MHC class II β strand. In some respects, the sequence encoding the MHC class II α strand is under the control of a first promoter, and the sequence encoding the MHC class II β strand is under the control of a second promoter.

[0319] In some respects, this disclosure relates to a first nucleic acid molecule encoding the MHC class II β chain disclosed herein and a second nucleic acid molecule encoding the MHC class II α chain disclosed herein.

[0320] Certain aspects of this disclosure relate to a vector or a group of vectors comprising the nucleic acid molecules 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.

[0321] In some respects, the vector is a retroviral vector. In some respects, the vector is an adenovirus vector, lentivirus, Sendai virus, baculovirus vector, Epstein-Barr virus vector, lactopolyvacuovirus vector, vaccinia virus vector, herpes simplex virus vector, or adeno-associated virus (AAV) vector. In certain respects, the vector is an AAV vector. In some respects, the vector is a lentivirus. In certain respects, the vector is an adenovirus vector. In some respects, the vector is Sendai virus. In some respects, the vector is a hybrid vector. Examples of hybrid vectors that may be used in this disclosure can be found in Huang and Kamihira, Biotechnol. Adv. 31(2):208-23(2103) (incorporated herein by reference in its entirety).

[0322] II.E. Methods of treating tumors

[0323] In some aspects, the methods disclosed herein also include treating cancer in a subject in need. In some aspects, the methods further include administering a TCR identified using the methods disclosed herein to a subject in need, wherein the subject has cancer. In some aspects, the methods include administering cells to the subject, wherein the cells contain a TCR identified using the methods disclosed herein. In some aspects, the cells are T cells.

[0324] In some respects, cancers include melanoma, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, malignant melanoma of the skin or eye, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, 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), esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, and adrenal cancer. Soft tissue sarcomas, urethral cancer, penile cancer, acute or chronic leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, including those induced by asbestos, environmentally induced cancers, other B-cell malignancies, and any combination of these cancers. In some respects, the cancer is melanoma.

[0325] In some ways, cancer is recurrent. In some ways, cancer is refractory. In some ways, cancer is advanced. In some ways, cancer is metastatic.

[0326] In some respects, the methods disclosed herein treat cancer in subjects. In some respects, the methods disclosed herein reduce the severity of one or more symptoms of cancer. In some respects, the methods disclosed herein reduce the size or number of tumors derived from said cancer. In some respects, the methods disclosed herein increase the overall survival of subjects compared to subjects not provided with the methods disclosed herein. In some respects, the methods disclosed herein increase the progression-free survival of subjects compared to subjects not provided with the methods disclosed herein. In some respects, the methods disclosed herein result in a partial response in subjects. In some respects, the methods disclosed herein result in a complete response in subjects.

[0327] Certain aspects of this disclosure relate to methods for treating infections in subjects in need, said methods comprising administering to the subject an HLA class II molecule disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, or a cell disclosed herein. Non-limiting examples of infections that can be treated using the compositions and methods disclosed herein include infections caused by viruses (including viroids and prions), bacteria, fungi, parasites, or any combination thereof. In some aspects, the virus is herpesvirus, HIV, papillomavirus, measles virus, rubella virus, human papillomavirus (HPV), human T-lymphovirus I, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, norovirus, and any combination thereof. In some aspects, the bacteria are selected from streptococci, staphylococci, and Escherichia coli. In some embodiments, bacterial infections are selected from brucellosis, Campylobacter infection, cat scratch disease, cholera, Escherichia coli infection, gonorrhea, Klebsiella infection, Enterobacter infection, Serratia infection, Legionella infection, meningococcal infection, pertussis, plague, Pseudomonas infection, Salmonella infection, Shigella infection, typhoid fever, tularemia, anthrax, diphtheria, enterococcal infection, erysipelothricosis, listeriosis, nocardiac infection, pneumococcal infection, staphylococcal infection, streptococcal infection, and any combination thereof. In some embodiments, parasitic infections are selected from pinworm infection, trichomoniasis, toxoplasmosis, giardiasis, cryptosporidiosis, malaria, hookworm infection, tinea, tapeworm infection, trematode infection, and any combination thereof. In some respects, fungal infections are selected from Candida, Malassezia furfur, dermatophytes (e.g., Epidermophyton, Microsporum, and Trichophyton), or any combination thereof.

[0328] In some respects, the methods disclosed herein include treating a subject with cancer or infection, the methods comprising administering to the subject cells described herein, wherein the cells comprise the MHC class II molecules disclosed herein, the nucleic acid molecules disclosed herein, the vectors disclosed herein, or any combination thereof.

[0329] In some cases, the cells are obtained from the subject. In other cases, the cells are obtained from donors other than the subject.

[0330] All aspects, facets, and options described in this article can be combined in any and all variations.

[0331] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference, as each individual publication, patent or patent application is specifically and individually indicated to the extent of its inclusion herein by reference.

[0332] This disclosure has been generally described, and further understanding can be obtained by referring to the embodiments provided herein. These embodiments are for illustrative purposes only and are not intended to be limiting.

[0333] Example

[0334] Example 1 – Generation of Affinity-Matured HLA-DP Molecules

[0335] cell

[0336] Peripheral mononuclear cells were obtained by density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare LifeSciences, Marlborough, MA). The K562 cell line is an erythroleukemia cell line with deficient HLA class I / II expression. K562-based artificial APCs (aAPCs) expressing various HLA class II genes as single HLA alleles along with CD80 and CD83 have been previously reported (Butler et al., PloS One 7, e30229 (2012)). The Jurkat 76 cell line is a T-cell leukemia cell line lacking endogenous TCR, CD4, and CD8 expression. Jurkat 76 / CD4 cells are generated by retroviral transduction of 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 (ThermoFisher Scientific, Waltham, MA). K562 and Jurkat 76 cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 50 μg / ml gentamicin.

[0337] peptides

[0338] The synthetic peptides were purchased from Genscript (Piscataway, NJ) and dissolved in DMSO at a concentration of 50 μg / ml. The peptide sequences are shown in Table 6.

[0339] Table 6: Synthetic peptide sequences

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347] Gene

[0348] Novel TCR genes were cloned using the SMARTer RACE 5' / 3' kit (Takara Bio, Shiga, Japan) via 5'-rapid amplification (RACE) PCR of the cDNA ends and sequenced as previously described. All genes were cloned into the pMX retroviral vector and transduced into cell lines using a retroviral system based on 293GPG and PG13 cells.

[0349] Antibody

[0350] The following antibodies were used for flow cytometry analysis: PE-conjugated anti-class II (9-49(I3)) and APC-Cy7-conjugated anti-CD4 (RPA-T4, Biolegend, San Diego, CA). 44 FITC-conjugated anti-NGFR (ME20.4, Biolegend, San Diego, CA), PE-conjugated anti-His tag (AD1.1.10, Abcam, Cambridge, MA), and FITC-conjugated anti-Vβ22 (IMMU 546, Beckman Coulter, Brea, CA). According to the manufacturer's instructions, PE-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA) was used to bind biotinylated DP4 / NY-ESO1. 157-170 and DP4 / WT1 329-348 Monomerization. Dead cells were distinguished using the Live / Dead Fixable Near-IR Dead Cell Stain Kit 465 (Thermo Fisher Scientific, Waltham, MA). Stained cells were analyzed using Canto II or LSR Tortessa X-20 (BD Biosciences, Franklin Lakes, NJ). Cell sorting was performed using FACS Aria II (BD Biosciences, Franklin Lakes, NJ). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR).

[0351] The following antibodies were used for immunoblotting analysis: anti-β-actin (C4, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-MAGE-A2 (Abcam, Cambridge, MA), anti-CCND1 (EPR2241, Abcam, Cambridge, MA), HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Promega, Fitchburg, WI), and HRP-conjugated anti-rabbit IgG (H+L) secondary antibody (Promega, Fitchburg, WI).

[0352] TCR transduction into primary T cells

[0353] Pan T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD4 were used respectively. + T-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) for purifying CD3 + and CD4 + T cells. Purified T cells were stimulated with 200 Gy of irradiated aAPC / mOKT3 at an E:T ratio of 20:1. Starting from the second day, activated T cells were transduced with a cloned TCR gene retrovirus by centrifugation at 1,000 × g for 1 hour at 32°C for 3 consecutive days or using a Retrotronectin-coated plate (Takara Bio, Shiga, Japan). On the second day, 100 IU / ml IL-2 and 10 ng / ml IL-15 were added to the TCR-transduced T cells. The culture medium was replenished every 2–3 days.

[0354] Stain with soluble CD4

[0355] The soluble CD4 (sCD4) gene is generated by fusing the extracellular domain of human CD4 with a 6xHis tag via a GS linker. HEK293T cells were transduced with an sCD4 gene retrovirus, and the culture supernatant containing sCD4 monomers was harvested. sCD4 was dimerized with PE-labeled anti-6xHis tag mAbs (AD1.1.10, Abcam, Cambridge, MA) and used. K562 cells expressing HLA class II were stained with dimerized sCD4 for 30 minutes at room temperature in the presence of goat serum. Surface HLA class II expression in K562-derived cells expressing various class II genes is shown in Figures 13A to 13Q.

[0356] Construction and screening of multi-site directed DPB1*04:01 mutant cDNA libraries

[0357] Multiple site-directed random mutations were inserted into the DPB1*04:01 cDNA using PCR and the following primer sets: For L112 and V114, forward: 5'-CACCACAACNNNCTTNNNTGCCACGTG-3' (SEQ ID NO:30) and reverse: 5'-CACGTGGCANNNAAGNNNGTTGTGGTG-3' (SEQ ID NO:31); For V141, forward: 5'-ACAGCTGGGGTCNNNTCCACCAACCTG-3' (SEQ ID NO:32) and reverse: 5'-CAGGTTGGTGGANNNGACCCCAGCTGT-3' (SEQ ID NO:33); For L156 and M158, forward: 5'-CAGATCNNNGTGNNNCTGGAAATGACC-3' (SEQ ID NO:34) and reverse: 5'-GGTCATTTCCAGNNNCACNNNGATCTG-3' (SEQ ID NO:35). N represents any nucleotide. The obtained PCR fragments were fused together to construct a mutant full-length DPB1*04:01 cDNA expression library, which carried random mutations at positions L112, V114, V141, L156, and M158. K562 cells stably expressing the DPA1*01:03 gene were infected with a recombinant retrovirus generated from the packaging cell line 293GPG at a transduction efficiency of less than 30%. Infected K562 cells were stained with soluble CD4 dimers, and dimer-positive cells were collected using flow cytometry cell sorting. The mutant DPB1*04:01 gene was cloned from the collected cells and, together with the wild-type DPA1*01:03 gene described above, was transduced into K562 cells via retrovirus.

[0358] Formation of HLA class II monomers and dimers

[0359] The extracellular domain of the wild-type class II α gene was fused to an acidic leucine zipper via a GGGS linker, followed by fusion to a 6xHis tag via a GS linker (see SEQ ID NO:8). The extracellular domain of the mutant class II β gene (see SEQ ID NO:3) was similarly linked to a basic leucine zipper via a GGGS linker (see SEQ ID NO:4). HEK293T cells were transfected with the α and β genes using a 293GPG cell-based retroviral system and cultured in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin. DP4 dimer staining was performed to ensure stable secretion of soluble DP4. L112W / V141MHEK293T cells containing the protein were grown to confluence, and the culture medium was replaced with serum-free 293 SFM II medium (Thermo Fisher Scientific, Waltham, MA). After 48 hours, the conditioned culture was harvested and concentrated using an Amicon ultrafilter (molecular weight cutoff (MWCO) 10 kDa) (Millipore Sigma, Burlington, MA). Then, the supernatant containing soluble HLA class II peptides was mixed with 100 μg / ml of the target peptide at 37°C for 20–24 hours for in vitro peptide exchange. Monomers that did not undergo peptide exchange served as controls. Monomer concentrations were measured by specific ELISA using nickel-coated plates (XPressBio, Frederick, MD) and anti-His-tagged biotinylated mAbs (AD1.1.10, R&D Systems, Minneapolis, MN). Soluble HLA class II monomers were dimerized at 4°C for 1.5 hours using PE-conjugated anti-HismAb (AD1.1.10, Abcam, Cambridge, MA) at a molar ratio of 2:1 for staining.

[0360] DP4-restricted antigen-specific CD4 + T cell stimulation

[0361] Using CD4 + T-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) for purifying CD4 + T cells. Purified T cells were stimulated with DP4-expressing aAPCs via a DP4-restricted peptide pulse at 10 μg / ml and irradiated at 200 Gy at an E:T ratio of 20:1. Forty-eight hours later, 10 IU / ml IL-2 and 10 ng / ml IL-15 were added to CD4. + In 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 / V141M Dimer staining.

[0362] HLA class II dimer and tetramer staining

[0363] Primary T cells transduced with the exogenous TCR gene and Jurkat 76 / CD4 T cells were pretreated with 50 nM dasatinib (LC Laboratories, Woburn, MA) at 37°C for 30 min, and then stained with class II dimers at 5-15 μg / ml for 4-5 h at room temperature. After washing, cell surface molecules were counterstained with APC-Cy7 conjugated anti-CD4 mAb, FITC conjugated anti-NGFR mAb, and PE conjugated anti-Vβ22 mAb.

[0364] ELISPOT measurement

[0365] As previously reported, the cytokine ELISPOT assay was performed (see, for example, Yamashita et al., Nat. Commun. 8:15244 (2017); and Anczurowski et al., Sci. Rep. 8:4804 (2018)).

[0366] Immunoblotting

[0367] As previously reported, immunoblotting analysis was performed (see, for example, Yamashita et al., Nat. Commun. 8:15244 (2017); and Anczurowski et al., Sci. Rep. 8:4804 (2018)).

[0368] Protein modeling

[0369] The structures of HLA-DP4 and human CD4 complex models were predicted using the Swiss-Model working area for quaternary structure prediction based on PDB IDs 3S5L and 3T0E.

[0370] Statistical analysis

[0371] Statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA). Unpaired two-tailed Student's t-tests were used for two-sample comparisons. No statistical methods were used to predetermine sample size. Researchers did not blind the allocations during the experiment or outcome evaluation. The experiment was not randomized.

[0372] Biological layer interferometric sensing image

[0373] The extracellular domain of human CD4, tagged with a GS linker and a 10x histidine (His) label (residues 26-440 of NP_000607.1), was stably expressed in the human cell line A375 (SEQ ID NOs: 262-263; Table 7). Recombinant 10x His-tagged CD4 protein was purified from the supernatant using TALON metal affinity resin (Takara Bio, Shiga, Japan). The eluted protein was concentrated using an Amicon Ultra-15 centrifuge column with a 10 kDa MWCO (MilliporeSigma, Burlington, MA). Buffer exchanged to HBS-EP (GE Healthcare Life Sciences, Marlborough, MA) was performed using a 10 kDa MWCO mini-dialyzer (Thermo Fisher Scientific, Waltham, MA). As confirmed by SDS-PAGE, the purity of the recombinant CD4 protein was consistently >90%.

[0374] The recombinant DP4 protein consists of the extracellular domain of DPA1*01:03 and either wild-type DPB1*04:01 or the L112W / V141M mutant. DPA1*01:03 is followed by an acidic leucine zipper, a GS linker, and a 10x histidine tag, while wild-type and mutant DPB1 are followed by a basic leucine zipper, a GS linker, and a biotinylated sequence (GLNDIFEAQKIEWHE; SEQ ID NO:265). Both DPA and DPB genes are stably expressed in A375-BirA cells, which were transduced using a BirA gene with codons optimized to encode a 5' leader sequence and a 3' ER-retained KDEL motif. The recombinant DP4 protein was purified from the supernatant using TALON metal affinity resin (Takara Bio, Shiga, Japan). The eluted proteins were concentrated using a Vivaspin 500 centrifuge column (GE Healthcare Life Sciences, Marlborough, MA) with a 10 kDa MWCO and restored to a working volume in PBS.

[0375] Wild-type DP4 and DP4 were measured using the Octet Red system (ForteBio, Fremont, CA). L112W / V141MBinding to CD4. Experiments were performed using 96-well OptiPlates (Perkin Elmer, Waltham, MA) at 25°C with a sample volume of 200 μl and continuous shaking at 1,000 rpm. Biotinylated recombinant DP4 was loaded onto streptavidin-coated biosensors (ForteBio, Fremont, CA) until saturation, followed by baseline measurements in HBS-EP buffer. Associativity was measured by incubating the loaded sensor with titrated concentrations of recombinant CD4 (0.8125 to 26 μM) for 400 seconds, followed by dissociation in separate HBS-EP buffer for 300 seconds. Steady-state analysis was performed using a single-point specific binding model fitted in GraphPad Prism 7.0.

[0376] Table 7. Soluble 10x His-labeled CD4 nucleic acid sequences

[0377]

[0378] Example 2 – L112W / V141M substitution of the DPβ chain enhances the binding of DP to CD4.

[0379] A cDNA expression library of the DPB1*04:01 (DP4β) gene carrying random mutations at L112, V114, V141, L156, and M158 (corresponding to L114, V116, V143, L158, and M160 of the DR1β chain, respectively) was generated and co-expressed with the wild-type DPA1*04:01 (DP4β) gene in class II defective K562 cells. After two rounds of selection using soluble CD4 protein (sCD4), a cell population with enhanced CD4 binding was isolated, from which the mutant DP4β gene carrying L112W, V114M, V141M, and M158I substitutions was molecularly cloned. When ectopically expressed in K562 cells, the wild-type DPα chain and the DP4β gene carrying L112W, V114M, V141M, and M158I substitutions were expressed. L112W / V114M / V141M / M158I The mutant DP4 molecule composed of the mutant DP4β chain of the clone did indeed show enhanced binding to sCD4 compared to the wild-type DP4 molecule, thus ruling out the possibility that the enhanced CD4 binding was an artificial product of the screening process (Figs. 1A to 1F).

[0380] To determine which of the four mutations was crucial for enhanced CD4 binding, reversion mutagenesis was performed. All possible reversion DP4 mutants were reconstructed in class II negative K562 cells and stained with sCD4. Both L112W and V141M, but not single substitutions of V114M or M158I, enhanced DP4 binding to sCD4. Figure 1GImportantly, the L112W / V141M double mutation (DP4) L112W / V141M ) Synergistically enhanced DP4 / CD4 binding ( Figure 1G Interestingly, both the V114M and M158I single-replacement displays support DP4. L112W / V141M Enhanced binding achieved through mutation has negative effects. Figure 1G Previous studies estimated the K-lineage between CD4 and HLA class II. D Value >2mM. DPR was measured using biolayer interferometry (BLI) combined with assay. L112 / V141M Affinity to CD4. Although no binding was detected between wild-type DP4 and CD4, DP4... L112W / V141M With K at 8.9 μM ± 1.1 D Combined with CD4 ( Figure 1H and Figure 1X to Figure 1BK This value indicates that binding affinity has increased by at least 200-fold. Furthermore, CD4 and DP4 were observed... L112W / V141M The affinity between DP4 and HLA class I is higher than that between human CD8 and HLA class I (approximately 200 μM), and comparable to that between mouse CD8 and mouse MHC class I (approximately 10 μM). To confirm DP4... L112W / V141M Enhanced binding between CD4 and CD4 results in enhanced CD4. + T-cell response, using CD4 transduced with DP4 / WT1 TCR (clone 9). - and CD4 + Jurkat 76 T cells act as responders against wild-type DP4 or DP4 expressing a single class II allele. L112W / V141M The immunostimulatory capacity of artificial APCs (aAPCs) was compared. As expected, those carrying DP4... L112W / V141M aAPCs exhibit enhanced T cell stimulation activity in a CD4-dependent manner. Figure 1I ).

[0381] Next, other DP alleles of CD4 were analyzed to determine whether the L112W / V141M mutation also enhanced binding. Although wild-type DP2, DP5, or DP8 do not bind to CD4, all three molecules showed strong binding to CD4 when the L112W / V141M double mutation was introduced into the DPβ chain of these molecules. Figure 1I (See Figure 1W). The structural model was constructed based on previously reported data. Figures 2A to 2D ) reveals that in DP4 L112W / V141MIn the CD4 complex, the two L112W / V141M mutations significantly induced hydrophobic effects at the K35, Q40, and T45 positions of CD4. These results indicate that the L112W / V141M mutation can enhance CD4 binding of at least all four tested DP alleles.

[0382] Example 3 – Affinity-matured DP4 L112W / V141M Multimer-specific staining homologous TCR

[0383] To determine the effect of the L112W / V141M double mutation of DP4β on DP4 multimer staining, soluble DP4 was produced. L112W / V141M Monomers were then dimerized with anti-His-tagged mAbs. Primary T cells were transduced with three different DP4-restricted TCRs specific to MAGE-A3 (clone R12C9), WT1 (clone 9), and NY-ESO-1 (clone 5B8), respectively, and then homologous DP4 was used. L112W / V141M二 Polymer staining. As shown in Figures 3A to 3P, each DP4... L112W / V141M Dimer-specific staining expresses CD4, a homologous TCR. + T cells. Anti-Vβ22 mAb and anti-NGFR mAb were administered along with their respective DP4 groups. L112W / V141M Complex staining of R12C9 and clone 9 transduced T cells with dimers confirmed that almost all TCR-transduced CD4+ cells were transduced. + T cells were successfully used with the corresponding DP4 L112W / V141M Dimer staining (Figs. 4A to 4H). Our novel DP4 tetramer, compared to the conventional wild-type DP4 tetramer... L112W / V141M The dimer showed superior staining performance on both DP4 / WT1 and DP4 / NY-ESO-1 T cells compared to conventional wild-type DP4 tetramer (Figs. 5A to 5P). Notably, even at the highest available concentration, conventional wild-type DP4 / NY-ESO-1 tetramer failed to stain homologous T cells (data not shown).

[0384] Example 4 – DP4 L112W / V141M Dimer technology is robust and versatile.

[0385] To prove DP4 L112W / V141M The robustness and versatility of multimer staining were demonstrated, and a comprehensive screening of the in vitro immunogenicity of potential DP4-restricted peptides derived from a range of tumor-associated antigens was conducted (Table 6). 196 DP4-restricted and tumor-associated antigen-derived 20-mer peptides were predicted using a peptide prediction algorithm (NetMHC2 ver. 2.2) and then chemically synthesized (Table 6). Peripheral antigen-specific CD4 + The frequency of T cells is generally very low; therefore, 6 DP4 cells will be selected.+ Primary CD4 isolated from melanoma patients + T cells were stimulated once with DP4-aAPC, and then pulsed with 196 peptides and homologous DP4. L112W / V141M Dimer staining. A weak stimulation condition was used to avoid potential in vitro priming. Figures 6A to 6F As shown, 103 predicted DP4 peptides are at least immunogenic in vitro.

[0386] To verify the dimer staining results, we cloned the inhibitor of CCND1 from dimer-positive T cells. 219-238 HSD17B12 225-244 LGSN 296-315 MAGE-A2 108-127 and MUC5AC 4922-4941 Seven DP4-specific TCR genes (Figures 7A to 7L and Table 8). When in human CD4... + During clonal remodeling in TCR-deficient T cells, all these TCRs are homologous to DP4. L112W / V141M The dimer was successfully stained (Figures 8A to 8X) and functioned in a DP4-restricted and antigen-specific manner. Figures 9A to 9G ).

[0387] Of the four TCRs expressed in primary T cells, three are O3-CCND1. 219-238 06-MAGE-A2 108-127 and 05-MUC5AC 4922-4941 It can recognize homologous peptides that are processed endogenously and presented by DP4 (Figure 10A to 10B). Figure 10Q and Figures 11A to 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. Figure 12A (See Figure 12E).

[0388] Table 8: DP4-Restricted TCR

[0389]

[0390]

[0391] Compared to CD8, the role and function of CD4 as a co-receptor are not fully elucidated. This lack of information is primarily due to the unusually weak binding between CD4 and class II receptors, which greatly limits research into the association between CD4 and class II receptors. In this study, the affinity-mature form of HLA-DP4, namely DP4, was isolated by enhanced CD4 binding. L112W / V141M And developed a new DP4L112W / V141M Dimer technology, which introduces the detection of DP4-restricted antigen-specific CD4. + The robustness and rigor of T cells.

[0392] Using this DP4 L112W / V141M Dimer technology was used to comprehensively study DP4-restricted anti-tumor T-cell responses in vitro and to identify multiple DP4-restricted immunogenic peptides and homologous TCR genes. HLA-DP4 is the most prevalent HLA allele in many ethnic groups and belongs to the DP4 family. 84Gly Group. Unlike other class II molecules, DP 84Gly Molecules such as DP4 constitutively present peptides of endogenous origin, regardless of constant chain and HLA-DM expression. Improved presentation of endogenous peptides via class II therapy has been associated with improved survival in cancer patients. Notably, the first-in-human class II restricted TCR gene therapy specifically targets the DP4-restricted MAGE-A3 peptide (see, for example, Yao et al., J. Immunother. 39:191-201 (2016)). 84Gly Genotypes such as DP2 and DP4 act as risk alleles for anti-neutrophil cytoplasmic autoantibody-associated vasculitis. The DP4 molecule can constitutively present peptides derived from endogenous tumor-associated antigens, and it may induce more clinically relevant antitumor responses than other class II molecules, thus acting as a protective class II allele.

[0393] To identify class II molecules with mature affinity, this embodiment details several mutations in the β chain rather than the α chain, because the β chain has a more direct interaction with CD4 than the α chain. Further mutations in the α and / or β chains may further enhance the binding between class II and CD4. However, using such soluble class II molecules with excessive CD4 binding capacity may lead to CD4... + Non-specific staining of T cells can have adverse effects.

[0394] In summary, CD4 + T cells play a crucial role in the development of autoimmune diseases and in protection against pathogen infection and cancer. The novel HLA class II multimer technology described in this article can better facilitate the HLA class II-restricted CD4 targeting of the HLA-DP allele. + Research on T cell responses.

[0395] Example 5 – Generation of Affinity-Matured HLA-DQ Molecules

[0396] cell

[0397] Peripheral mononuclear cells (Ficoll-Paque PLUS, GE Healthcare LifeSciences, Marlborough, MA) were obtained by density gradient centrifugation. The K562 cell line is an erythroleukemia cell line with defective HLA class I / II expression. A375 is a melanoma cell line. HEK293T cells and A375 cells were grown in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin (Thermo Fisher Scientific, Waltham, MA). K562 and Jurkat 76 cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 50 μg / ml gentamicin.

[0398] peptides

[0399] The synthetic peptides were purchased from Genscript (Piscataway, NJ) and dissolved in DMSO at a concentration of 50 μg / ml.

[0400] Antibody

[0401] The following antibodies were used for flow cytometry analysis: PE-conjugated anti-class II (9-49(I3), Beckman Coulter, Brea, CA; Tü39), APC-Cy7-conjugated anti-CD4 (RPA-T4, Biolegend, San Diego, CA), and PE-conjugated anti-His tag (AD1.1.10, Abcam, Cambridge, MA). Dead cells were distinguished using the Live / Dead Fixable Near-Infrared Dead Cell Staining Kit 465 (Thermo Fisher Scientific, Waltham, MA). Stained cells were analyzed using Canto II or LSR Fretessa X-20 (BD Biosciences, Franklin Lakes, NJ). Cell sorting was performed using FACS Aria II (BD Biosciences, Franklin Lakes, NJ). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR).

[0402] TCR transduction into primary T cells

[0403] Pan T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD4 were used respectively. + T-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) for purifying CD3 +and CD4 + T cells. Purified T cells were stimulated with 200 Gy of irradiated aAPC / mOKT3 at an E:T ratio of 20:1. Starting from the second day, activated T cells were transduced with a cloned TCR gene retrovirus by centrifugation at 1,000 × g for 1 hour at 32°C for 3 consecutive days or using a Retrotronectin-coated plate (Takara Bio, Shiga, Japan). On the second day, 100 IU / ml IL-2 and 10 ng / ml IL-15 were added to the TCR-transduced T cells. The culture medium was replenished every 2–3 days.

[0404] Stain with soluble CD4

[0405] The soluble CD4 (sCD4) gene is generated by fusing the extracellular domain of human CD4 with a 6xHis tag via a GS linker. HEK293T cells were transduced with an sCD4 gene retrovirus, and the culture supernatant containing sCD4 monomers was harvested. sCD4 was dimerized with PE-labeled anti-6xHis tag mAbs (AD1.1.10, Abcam, Cambridge, MA) and used. K562 cells expressing HLA class II were stained with dimerized sCD4 for 30 minutes at room temperature in the presence of goat serum. Surface HLA class II expression in K562-derived cells expressing various class II genes is shown in Figures 16A to 16Q.

[0406] Formation of HLA class II monomers and dimers

[0407] The extracellular domain of the wild-type class II α gene was fused to an acidic leucine zipper via a GGGS linker, followed by fusion to a 6xHis tag via a GS linker (see SEQ ID NO:18). The extracellular domain of the mutant class II β gene (see SEQ ID NO:13) was similarly linked to a basic leucine zipper via a GGGS linker (see SEQ ID NO:14). HEK293T and A375 cells were transfected with the α and β genes using a 293GPG cell-based retroviral system and cultured in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin. For dimer staining, stable secretion of soluble DQ5 was achieved. L114W / V143M+4reps (In addition to L114W / V143M, it also has N110Q / I116V / S118H / P146N replacements (4 reps)) and DQ6 L114W / V143M+3repsA375 cells (containing N110Q / S118H / P146N replacements (3 reps) in addition to L114W / V143M) were grown to confluence, and the culture medium was harvested after 48 hours. In vitro peptide exchange was then performed by mixing the supernatant containing soluble HLA class II with 100 μg / ml of the target peptide at 37°C for 20–24 hours. Monomers not subjected to peptide exchange served as controls. Monomer concentrations were measured by specific ELISA using nickel-coated plates (XPressBio, Frederick, MD) and an anti-His-tagged biotinylated mAb (AD1.1.10, R&D Systems, Minneapolis, MN). Soluble HLA class II monomers were dimerized at 4°C for 1.5 hours using a PE-conjugated anti-His mAb (AD1.1.10, Abcam, Cambridge, MA) at a 2:1 molar ratio for staining.

[0408] HLA class II dimer staining

[0409] Primary T cells transduced with the exogenous TCR gene were pretreated with 50 nM dasatinib (LC Laboratories, Woburn, MA) at 37°C for 30 min, and then stained with class II dimers at 5-15 μg / ml for 4-5 h at room temperature. After washing, cell surface molecules were counterstained with APC-Cy7 conjugated anti-CD4 mAb.

[0410] Statistical analysis

[0411] Statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA). Unpaired two-tailed Student's t-tests were used for two-sample comparisons. No statistical methods were used to predetermine sample size. Researchers did not blind the allocations during the experiment or outcome evaluation. The experiment was not randomized.

[0412] Example 6 – DQ molecule with enhanced CD4 binding ability

[0413] By introducing the L114W / V143M mutation to generate DQ molecules with enhanced affinity, we determined whether these substitutions could improve the binding of HLA-DQ molecules such as DQ5 (DQA1*01:01-DQB1*05:01) to CD4. In addition to positions 114 and 143, DQB1*05:01 also encodes four different amino acids at positions 110, 116, 118, and 146. Therefore, we generated expression of DQ5... L114W / V143M+4reps K562 cells, except for L114W / V143M ( Figure 14AIn addition, it also possesses N110Q / I116V / S118H / P146N substitutions (4 reps), and these cells were stained with sCD4. It expresses DQ5. L114W / V143M+4reps But not expressing DQ5 L114W / V143M DQ5 4reps K562 cells with wild-type DQ5 showed enhanced CD4 binding (or wild-type DQ5 cells showed enhanced CD4 binding). Figures 14B to 14C Importantly, various DQ5 molecules, each expressed at one of the four positions, exhibit a single amino acid inversion. L114W / V143M+4reps A series of K562 cells from mutant strains lacked enhanced CD4 binding ability. Figure 14D These results indicate that the four additional substitutions at N110Q, I116V, S118H, and P146N are crucial for the effectiveness of the L114W / V143M mutation in the observed enhanced DQ5:CD4 binding.

[0414] DQβ chains such as DQB1*02:01, 04:02, and 06:01 encode different amino acids at positions 110, 118, and 146, rather than at position 116. Figure 14E Unlike DQB1*05:01, DQB1*02:01, 04:02, and 06:01 encode Val at position 116, similar to DPB1*04:01, which encodes Val at position 114. All DQ2... L114W / V143M+3reps DQ4 L114W / V143M+3reps and DQ6 L114W / V143M+3reps The mutant (whose β chain carries N110Q, S118H, and P146N substitutions (3 reps) along with L114W / V143M) exhibits enhanced CD4 binding activity. Figure 14F ).

[0415] Example 7 – Affinity-matured DQ dimer specificity and robust staining homologous TCR

[0416] The affinity maturation of DQ dimers carrying the mutations described in Example 2 was evaluated to identify antigen-specific CD4. + The ability of T cells. DQ5 L114W / V143M+4reps and DQ6 L114W / V143M+3reps The dimer successfully stained DQ5-restricted DDX3Y-specific TCR (E6) and DQ6-restricted influenza virus-specific TCR (DM2), respectively. Figures 15A to 15B ).

[0417] To identify class II molecules with mature affinity, this embodiment details several mutations in the β chain rather than the α chain, because the β chain has a more direct interaction with CD4 than the α chain. Further mutations in the α and / or β chains may further enhance the binding between class II and CD4. However, using such soluble class II molecules with excessive CD4 binding capacity may lead to CD4... + Non-specific staining of T cells can have adverse effects.

[0418] In summary, CD4 + T cells play a crucial role in the development of autoimmune diseases and in protection against pathogen infection and cancer. The novel HLA class II multimer technology described in this article can better facilitate the HLA class II-restricted CD4 targeting of the HLA-DQ allele. + Research on T cell responses.

[0419] Example 8 – Generation of Affinity-Matured HLA-DR Molecules

[0420] cell

[0421] Peripheral mononuclear cells were obtained by density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare LifeSciences, Marlborough, MA). The K562 cell line is an erythroleukemia cell line with defective HLA class I / II expression. K562-based artificial APCs (aAPCs) expressing various HLA class II genes as single HLA alleles along with CD80 and CD83 have been previously reported (Butler et al., PloS One 7, e30229 (2012)). HEK293T cells were grown in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin (Thermo Fisher Scientific, Waltham, MA). K562 cells were cultured in RPMI 1640 supplemented with 10% FBS and 50 μg / ml gentamicin.

[0422] peptides

[0423] The synthetic peptides were purchased from Genscript (Piscataway, NJ) and dissolved in DMSO at a concentration of 50 μg / ml.

[0424] Antibody

[0425] The following antibodies were used for flow cytometry analysis: PE-conjugated anti-class II (9-49(I3)) and APC-Cy7-conjugated anti-CD4 (RPA-T4, Biolegend, San Diego, CA). 44PE-conjugated anti-His tags (AD1.1.10, Abcam, Cambridge, MA) and FITC-conjugated anti-Vβ22 tags (IMMU 546, Beckman Coulter, Brea, CA) were used. Dead cells were distinguished using the live / dead fixable near-infrared dead cell staining kit 465 (Thermo Fisher Scientific, Waltham, MA). Stained cells were analyzed using Canto II or LSR Tortessa X-20 (BD Biosciences, Franklin Lakes, NJ). Cell sorting was performed using FACS Aria II (BD Biosciences, Franklin Lakes, NJ). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR).

[0426] TCR transduction into primary T cells

[0427] Using CD4 + T-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) for purifying CD4 + T cells. Purified T cells were stimulated with 200 Gy of irradiated aAPC / mOKT3 at an E:T ratio of 20:1. Starting from the second day, activated T cells were transduced with a cloned TCR gene retrovirus by centrifugation at 1,000 × g for 1 hour at 32°C for 3 consecutive days or using a Retrotronectin-coated plate (Takara Bio, Shiga, Japan). On the second day, 100 IU / ml IL-2 and 10 ng / ml IL-15 were added to the TCR-transduced T cells. The culture...

Claims

1. A method for identifying MHC class II specific T cell receptors, the method comprising contacting T cells with a complex comprising MHC class II molecules and peptides; The T cells expressed CD4 and one or more T cell receptors; The MHC class II molecules described herein comprise DP α chains and DP β chains, wherein, The amino acid sequence of the extracellular domain of the DP β chain is shown in SEQ ID NO: 3; The MHC class II molecules have a higher affinity for CD4 than naturally occurring MHC class II molecules; and The MHC class II specific T cell receptor-specific binding described therein comprises a complex of the MHC class II molecule and the peptide.

2. The method of claim 1, wherein the DP β chain of the MHC class II molecule is encoded by alleles of DP1, DP2, DP3, DP4, DP5, DP6, DP8 or DP9.

3. The method of claim 1, wherein the DPβ chain of the MHC class II molecule is encoded by 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*108, DPB1*109, DPB1*110, DPB1*111, DPB1*112, DPB1*113, DPB1*114, DPB1*115, DPB1*116, DPB1*117, DPB1*118, DPB1*1 ... 1*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* D PB1*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*1 66. 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, DP B1*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 alleles.

4. The method of claim 1, wherein the DPα chain of the MHC class II molecule contains the HLA-DPA1*01, HLA-DPA1*02, HLA-DPA1*03 or HLA-DPA1*04 alleles.

5. A method for recognizing MHC class II specific T cell receptors, including contacting T cells with a complex containing MHC class II molecules and peptides; The T cells described therein express CD4 and one or more T cell receptors; The MHC class II molecule comprises a DQ α chain and a DQ β chain, wherein the amino acid sequence of the extracellular domain of the DQ β chain is shown in SEQ ID NO: 13; The MHC class II molecules have a higher affinity for CD4 than naturally occurring MHC class II molecules; and The MHC class II specific T cell receptor specifically binds to the complex containing MHC class II molecules and peptides.

6. The method of claim 5, wherein the DQ β chain of the MHC class II molecule is encoded by the DQ2, DQ3, DQ4, DQ5 or DQ6 alleles.

7. The method of claim 5, wherein the DQ β chain of the MHC class II molecule is encoded by an allele of HLA-DQB1*02, HLA-DQB1*03, HLA-DQB1*04, HLA-DQB1*05 or HLA-DQB1*06.

8. The method of claim 5, wherein the DQ α chain of the MHC class II molecule is encoded by the HLA-DQA1*01, HLA-DQA1*02, HLA-DQA1*03, HLA-DQA1*04, HLA-DQA1*05 or HLA-DQA1*06 alleles.

9. A method for recognizing MHC class II specific T cell receptors, comprising contacting T cells with a complex comprising MHC class II molecules and peptides; The T cells described therein express CD4 and one or more T cell receptors; The MHC class II molecule comprises a DR α chain and a DR β chain, wherein the amino acid sequence of the extracellular domain of the DR β chain is shown in SEQ ID NO:

21. The MHC class II molecules have a higher affinity for CD4 than naturally occurring MHC class II molecules; and The MHC class II specific T cell receptor specifically binds to the complex containing MHC class II molecules and peptides.

10. The method of claim 9, wherein the DR β chain of the MHC class II molecule is encoded by alleles of DR2, DR3, DR4, DR5, DR6, DR7, DR8, DR9, DR10, DR11, DR12, DR13, DR14, DR15 or DR16.

11. The method of claim 9, wherein the DR β chain of the MHC class II molecule is encoded by 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.

12. The method of claim 9, wherein the DR α chain of the MHC class II molecule is encoded by the HLA-DRA1*01 allele.

13. The method of any one of claims 1 to 12, wherein the MHC class II molecule is a dimer.

14. The method of any one of claims 1 to 12, wherein the MHC class II molecule is a trimer.

15. The method of any one of claims 1 to 12, wherein the MHC class II molecule is a tetramer.

16. The method of any one of claims 1 to 12, wherein the peptide comprises a fragment of a protein.

17. The method of claim 16, wherein the protein is expressed by diseased cells.

18. The method of claim 16, wherein the protein is expressed by tumor cells.

19. The method of any one of claims 1 to 12, wherein the peptide comprises at least 10 amino acids.

20. The method of claim 19, wherein the peptide comprises 10 to 100 amino acids.

21. The method of claim 19, wherein the peptide comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids.

22. The method of any one of claims 1-12, wherein the MHC class II molecules are expressed on the surface of antigen-presenting cells.

23. The method of any one of claims 1-12, wherein the T cells are obtained from a human subject.

24. The method of any one of claims 1-12, wherein the T cells are tumor-infiltrating lymphocytes (TILs).

25. The method of any one of claims 1 to 12, wherein the affinity of the MHC class II molecule for CD4 is at least twice that of the binding affinity of naturally occurring MHC class II molecules for CD4.

26. The method of any one of claims 1 to 12, the method further comprising selecting T cells that are bound by the MHC class II molecule.

27. The method of any one of claims 1 to 12, further comprising isolating T-cell receptors that bind to the MHC class II molecule.

28. The method of claim 27, further comprising sequencing the T cell receptor.

29. The method of claim 27, further comprising cloning the T cell receptor.

30. The method of claim 27, further comprising recombinantly expressing the T cell receptor in a host cell.

31. The method according to any one of claims 1 to 12, wherein the MHC class II molecules are in a K+ concentration of less than 100 μM. D Combined with CD4.

32. The method of any one of claims 1 to 12, wherein the MHC class II molecules are at 14 μM or less K D Combined with CD4.

33. The method of any one of claims 1 to 12, wherein the MHC class II molecules are at 8.9 μM or less K D Combined with CD4.

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