MHC class II molecules and methods of use thereof

Abstract

The present disclosure relates to HLA class II molecules that have a higher affinity for CD4 than naturally occurring HLA class II molecules. In certain embodiments, the HLA class II molecules comprise a DP beta chain having (i) an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1, (ii) an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1, or (iii) both (i) and (ii). Certain embodiments of the present disclosure relate to nucleic acid molecules encoding the HLA class II molecules, vectors comprising the nucleic acid molecules, cells comprising the vectors, and methods of use thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of U.S. Provisional Patent Application No. 62 / 880,496, filed July 30, 2019, and U.S. Provisional Patent Application No. 63 / 029,111, filed May 22, 2020, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Reference to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted Sequence Listing (Name: 4285.010PC02_SL_ST25.txt, Size: 144,830 bytes; and Created: July 28, 2020) are incorporated herein by reference in their entirety.

[0003] The present disclosure provides major histocompatibility complex (MHC) class II molecules with enhanced affinity for CD4, and methods of use thereof. [Background technology]

[0004] Immunotherapy has emerged as an important tool in the fight against various diseases, including cancer. T cell therapy is at the forefront of immunotherapeutic development, and adoptive transfer of antitumor T cells has been shown to induce clinical responses in cancer patients.

[0005] Targeted T cell therapy, which uses T cells expressing T cell receptors (TCRs) specific for target epitopes expressed on tumor cells, is a promising form of T cell therapy. Antigen-presenting cells (APCs) induce immune responses by presenting peptide fragments associated with major histocompatibility complex (MHC) receptors on their surface. Improved presentation of endogenous peptides by MHC class II has previously been demonstrated to correlate with prolonged survival in cancer patients. However, the development of novel TCRs that can specifically target peptides presented by MHC class II is limited by the low affinity of MHC class II proteins for CD4 expressed on T cells.

[0006] The present disclosure provides MHC class II proteins with enhanced affinity for CD4 and methods of using same for the identification and improvement of novel MHC class II-specific TCRs. Summary of the Invention [Means for solving the problem]

[0007] Certain aspects of the present disclosure relate to HLA class II molecules comprising a DP beta chain, wherein the DP beta chain comprises an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO:1.

[0008] Certain aspects of the present disclosure relate to an HLA class II molecule comprising a DP beta chain, wherein the DP beta chain comprises a substitution mutation at an amino acid residue corresponding to position 112 of SEQ ID NO: 1, wherein the substitution mutation is with an amino acid other than leucine.

[0009] In some embodiments, the DP beta chain further comprises an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO:1.

[0010] Certain aspects of the present disclosure relate to HLA class II molecules comprising a DP beta chain, wherein the DP beta chain comprises an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO:1.

[0011] Certain aspects of the present disclosure relate to an HLA class II molecule comprising a DP beta chain, wherein the DP beta chain comprises a substitution mutation at an amino acid residue corresponding to position 141 of SEQ ID NO: 1, wherein the substitution mutation is with an amino acid other than valine.

[0012] In some embodiments, the DP beta chain further comprises an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO:1.

[0013] In some embodiments, the DP beta chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 3, 4, and 5.

[0014] In some embodiments, the amino acid other than leucine comprises a hydrophobic side chain.

[0015] In some embodiments, the amino acid other than leucine is selected from the group consisting of alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0016] In some embodiments, the amino acid other than leucine is tryptophan.

[0017] In some embodiments, the amino acid other than valine comprises a hydrophobic side chain. In some embodiments, the amino acid other than valine is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the amino acid other than valine is methionine.

[0018] In some embodiments, the DP beta chain comprises a tryptophan at the amino acid residue corresponding to position 112 of SEQ ID NO:1 and a methionine at the amino acid residue corresponding to position 141 of SEQ ID NO:1.

[0019] In some embodiments, the DP beta chain comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the DP beta chain comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0020] In some embodiments, the DP beta chain is 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*11, DPB1*110, DPB1*111, DPB1*112, DPB1*113, DPB1*114, DPB1*115, DPB1*1 *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, DP B1*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, DP B1*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, D PB1*155, DPB1*156, DPB1*157, DPB1*158, DPB1*159, DPB1*16, DPB1*160, DPB1*161, DPB1*162, DPB1*163, DPB1*164, DPB1*165, DPB1*166, DPB1*167, DPB1*168, DPB1*169, DPB1*17, DPB1*170, DPB1*171, DPB1*172, DPB1*173, DPB1*174, DPB1*175, DPB1*176, DPB1*177, DPB1*178, DPB1*179, DPB1*18, DPB1*180, DPB1*181, DPB1*182, DPB1*183, DPB1*184, DPB1*185, DPB1*186, DPB1*187, DPB1*188, DPB1*189, DPB1*19, DPB1*190, DPB1*191, DPB1*192,DPB1*193、DPB1*194、DPB1*195、DPB1*196、DPB1*197、DPB1*198、DPB1*199、DPB1*20、DPB1*200、DPB1*201、DPB1*202、DPB1*203、DPB1*204、DPB1*205、DPB1*206、DPB1*207、DPB1*208、DPB1*209、DPB1*21、DPB1*210、DPB1*211、DPB1*212、DPB1*213、DPB1*214、DPB1*215、DPB1*216、DPB1*217、DPB1*218、DPB1*219、DPB1*22、DPB1*220、DPB1*221、DPB1*222、DPB1*223、DPB1*224、DPB1*225、DPB1*226、DPB1*227、DPB1*228、DPB1*229、DPB1*23、DPB1*230、DPB1*231、DPB1*232、DPB1*233、DPB1*234、DPB1*235、DPB1*236、DPB1*237、DPB1*238、DPB1*239、DPB1*24、DPB1*240、DPB1*241、DPB1*242、DPB1*243、DPB1*244、DPB1*245、DPB1*246、DPB1*247、DPB1*248、DPB1*249、DPB1*25、DPB1*250、DPB1*251、DPB1*252、DPB1*253、DPB1*254、DPB1*255、DPB1*256、DPB1*257、DPB1*258、DPB1*259、DPB1*26、DPB1*260、DPB1*261、DPB1*262、DPB1*263、DPB1*264、DPB1*265、DPB1*266、DPB1*267、DPB1*268、DPB1*269、DPB1*27、DPB1*270、DPB1*271、DPB1*272、DPB1*273、DPB1*274、DPB1*275、DPB1*276、DPB1*277、DPB1*278、DPB1*279、DPB1*28、DPB1*280、DPB1*281、DPB1*282、DPB1*283、DPB1*284、DPB1*285、DPB1*286、DPB1*287、DPB1*288、DPB1*289、DPB1*29、DPB1*290、DPB1*291、DPB1*292、DPB1*293、DPB1*294、DPB1*295、DPB1*296、DPB1*297、DPB1*298、DPB1*299、DPB1*30、DPB1*300、DPB1*301、DPB1*302、DPB1*303、DPB1*304、DPB1*305、DPB1*306、DPB1*307、DPB1*308、DPB1*309、DPB1*31、DPB1*310、DPB1*311、DPB1*312、DPB1*313、DPB1*314、DPB1*315、DPB1*316、DPB1*317、DPB1*318、DPB1*319、DPB1*32、DPB1*320、DPB1*321、DPB1*322、DPB1*323、DPB1*324、DPB1*325、DPB1*326、DPB1*327、DPB1*328、DPB1*329、DPB1*33、DPB1*330、DPB1*331、DPB1*332、DPB1*333、DPB1*334、DPB1*335、DPB1*336、DPB1*337、DPB1*338、DPB1*339、DPB1*34、DPB1*340、DPB1*341、DPB1*342、DPB1*343、DPB1*344、DPB1*345、DPB1*346、DPB1*347、DPB1*348、DPB1*349、DPB1*35、DPB1*350、DPB1*351、DPB1*352、DPB1*353、DPB1*354、DPB1*355、DPB1*356、DPB1*357、DPB1*358、DPB1*359、DPB1*36、DPB1*360、DPB1*361、DPB1*362、DPB1*363、DPB1*364、DPB1*365、DPB1*366、DPB1*367、DPB1*368、DPB1*369、DPB1*37、DPB1*370、DPB1*371、DPB1*372、DPB1*373、DPB1*374、DPB1*375、DPB1*376、DPB1*377、DPB1*378、DPB1*379、DPB1*38、DPB1*380、DPB1*381、DPB1*382、DPB1*383、DPB1*384、DPB1*385、DPB1*386、DPB1*387、DPB1*388、DPB1*389、DPB1*39、DPB1*390、DPB1*391、DPB1*392、DPB1*393、DPB1*394、DPB1*395、DPB1*396、DPB1*397、DPB1*398、DPB1*399、DPB1*40、DPB1*400、DPB1*401、DPB1*402、DPB1*403、DPB1*404、DPB1*405、DPB1*406、DPB1*407、DPB1*408、DPB1*409、DPB1*41、DPB1*410、DPB1*411、DPB1*412、DPB1*413、DPB1*414、DPB1*415、DPB1*416、DPB1*417、DPB1*418、DPB1*419、DPB1*420、DPB1*421、DPB1*422、DPB1*423、DPB1*424、DPB1*425、DPB1*426、DPB1*427、DPB1*428、DPB1*429、DPB1*430、DPB1*431、DPB1*432、DPB1*433、DPB1*434、DPB1*435、DPB1*436、DPB1*437、DPB1*438、DPB1*439、DPB1*44、DPB1*440、DPB1*441、DPB1*442、DPB1*443、DPB1*444、DPB1*445、DPB1*446、DPB1*447、DPB1*448、DPB1*449、DPB1*45、DPB1*450、DPB1*451、DPB1*452、DPB1*453、DPB1*454、DPB1*455、DPB1*456、DPB1*457、DPB1*458、DPB1*459、DPB1*46、DPB1*460、DPB1*461、DPB1*462、DPB1*463、DPB1*464、DPB1*465、DPB1*466、DPB1*467、DPB1*468、DPB1*469、DPB1*47、DPB1*470、DPB1*471、DPB1*472、DPB1*473、DPB1*474、DPB1*475、DPB1*476、DPB1*477、DPB1*478、DPB1*479、DPB1*48、DPB1*480、DPB1*481、DPB1*482、DPB1*483、DPB1*484、DPB1*485、DPB1*486、DPB1*487、DPB1*488、DPB1*489、DPB1*49、DPB1*490、DPB1*491、DPB1*492、DPB1*493、DPB1*494、DPB1*495、DPB1*496、DPB1*497、DPB1*498、DPB1*499、DPB1*50、DPB1*500、DPB1*501、DPB1*502、DPB1*503、DPB1*504、DPB1*505、DPB1*506、DPB1*507、DPB1*508、DPB1*509、DPB1*51、DPB1*510、DPB1*511、DPB1*512、DPB1*513、DPB1*514、DPB1*515、DPB1*516、DPB1*517、DPB1*518、DPB1*519、DPB1*52、DPB1*520、DPB1*521、DPB1*522、DPB1*523、DPB1*524、DPB1*525、DPB1*526、DPB1*527、DPB1*528、DPB1*529、DPB1*53、DPB1*530、DPB1*531、DPB1*532、DPB1*533、DPB1*534、DPB1*535、DPB1*536、DPB1*537、DPB1*538、DPB1*539、DPB1*54、DPB1*540、DPB1*541、DPB1*542、DPB1*543、DPB1*544、DPB1*545、DPB1*546、DPB1*547、DPB1*548、DPB1*549、DPB1*55、DPB1*550、DPB1*551、DPB1*552、DPB1*553、DPB1*554、DPB1*555、DPB1*556、DPB1*557、DPB1*558、DPB1*559、DPB1*56、DPB1*560、DPB1*561、DPB1*562、DPB1*563、DPB1*564、DPB1*565、DPB1*566、DPB1*567、DPB1*568、DPB1*569、DPB1*57、DPB1*570、DPB1*571、DPB1*572、DPB1*573、DPB1*574、DPB1*575、DPB1*576、DPB1*577、DPB1*578、DPB1*579、DPB1*58、DPB1*580、DPB1*581、DPB1*582、DPB1*583、DPB1*584、DPB1*585、DPB1*586、DPB1*587、DPB1*588、DPB1*589、DPB1*59、DPB1*590、DPB1*591、DPB1*592、DPB1*593、DPB1*594、DPB1*595、DPB1*596、DPB1*597、DPB1*598、DPB1*599、DPB1*60、DPB1*600、DPB1*601、DPB1*602、DPB1*603、DPB1*604、DPB1*605、DPB1*606、DPB1*607、DPB1*608、DPB1*609、DPB1*61、DPB1*610、DPB1*611、DPB1*612、DPB1*613、DPB1*614、DPB1*615、 DPB1*616、DPB1*617、DPB1*618、DPB1*619、DPB1*62、DPB1*620、DPB1*621、DPB1*622、DPB1*623、DPB1*624、DPB1*625、DPB1*626、DPB1*627、DPB1*628、DPB1*629、DPB1*63、DPB1*630、DPB1*631、DPB1*632、DPB1*633、DPB1*634、DPB1*635、DPB1*636、DPB1*637、DPB1*638、DPB1*639、DPB1*64、DPB1*640、DPB1*641、DPB1*642、DPB1*643、DPB1*644、DPB1*645、DPB1*646、DPB1*647、DPB1*648、DPB1*649、DPB1*65、DPB1*650、DPB1*651、DPB1*652、DPB1*653、DPB1*654、DPB1*655、DPB1*656、DPB1*657、DPB1*658、DPB1*659、DPB1*66、DPB1*660、DPB1*661、DPB1*662、DPB1*663、DPB1*664、DPB1*665、DPB1*666、DPB1*667、DPB1*668、DPB1*669、DPB1*67、DPB1*670、DPB1*671、DPB1*672、DPB1*673、DPB1*674、DPB1*675、DPB1*676、DPB1*677、DPB1*678、DPB1*679、DPB1*68、DPB1*680、DPB1*681、DPB1*682、DPB1*683、DPB1*684、DPB1*685、DPB1*686、DPB1*687、DPB1*688、DPB1*689、DPB1*69、DPB1*690、DPB1*691、DPB1*692、DPB1*693、DPB1*694、DPB1*695、DPB1*696、DPB1*697、DPB1*698、DPB1*699、DPB1*70、DPB1*700、DPB1*701、DPB1*702、DPB1*703、DPB1*704、DPB1*705、DPB1*706、DPB1*707、DPB1*708、DPB1*709、DPB1*71、DPB1*710、DPB1*711、DPB1*712、DPB1*713、DPB1*714、DPB1*715、DPB1*716、DPB1*717、DPB1*718、DPB1*719、DPB1*72、DPB1*720、DPB1*721、DPB1*722、DPB1*723、DPB1*724、DPB1*725、DPB1*726、DPB1*727、DPB1*728、DPB1*729、DPB1*73、DPB1*730、DPB1*731、DPB1*732、DPB1*733、DPB1*734、DPB1*735、DPB1*736、DPB1*737、DPB1*738、DPB1*739、DPB1*74、DPB1*740、DPB1*741、DPB1*742、DPB1*743、DPB1*744、DPB1*745、DPB1*746、DPB1*747、DPB1*748、DPB1*749、DPB1*75、DPB1*750、DPB1*751、DPB1*752、DPB1*753、DPB1*754、DPB1*755、DPB1*756、DPB1*757、DPB1*758、DPB1*759、DPB1*76、DPB1*760、DPB1*761、DPB1*762、DPB1*763、DPB1*764、DPB1*765、DPB1*766、DPB1*767、DPB1*768、DPB1*769、DPB1*77、DPB1*770、DPB1*771、DPB1*772、DPB1*773、DPB1*774、DPB1*775、DPB1*776、DPB1*777、DPB1*778、DPB1*779、DPB1*78、DPB1*780、DPB1*781、DPB1*782、DPB1*783、DPB1*784、DPB1*785、DPB1*786、DPB1*787、DPB1*788、DPB1*789、DPB1*79、DPB1*790、DPB1*791、DPB1*792、DPB1*794、DPB1*795、DPB1*796、DPB1*797、DPB1*798、DPB1*799、DPB1*80、DPB1*800、DPB1*801、DPB1*802、DPB1*803、DPB1*804、DPB1*805、DPB1*806、DPB1*807、DPB1*808、DPB1*809、DPB1*81、DPB1*810、DPB1*811、DPB1*812、DPB1*813、DPB1*814、DPB1*815、DPB1*816、DPB1*817、DPB1*818、DPB1*819、DPB1*82、DPB1*820、DPB1*821、DPB1*822、DPB1*823、DPB1*824、DPB1*825、DPB1*826、DPB1*827、DPB1*828、DPB1*829、DPB1*83、DPB1*830、DPB1*831、DPB1*832、DPB1*833、DPB1*834、DPB1*835、DPB1*836、DPB1*837、DPB1*838、DPB1*839、DPB1*84、DPB1*840、DPB1*841、DPB1*842、DPB1*843、DPB1*844、DPB1*845、DPB1*846、DPB1*847、DPB1*848、DPB1*849、DPB1*85、DPB1*850、DPB1*851、DPB1*852、DPB1*853、DPB1*854、DPB1*855、DPB1*856、DPB1*857、DPB1*858、DPB1*859、DPB1*86、DPB1*860、DPB1*861、DPB1*862、DPB1*863、DPB1*864、DPB1*865、DPB1*866、DPB1*867、DPB1*868、DPB1*869、DPB1*87、DPB1*870、DPB1*871、DPB1*872、DPB1*873、DPB1*874、DPB1*875、DPB1*876、DPB1*877、DPB1*878、DPB1*879、DPB1*88、DPB1*880、DPB1*881、DPB1*882、DPB1*883、DPB1*884、DPB1*885、DPB1*886、DPB1*887、DPB1*888、DPB1*889、DPB1*89、DPB1*890、DPB1*891、DPB1*892、DPB1*893、DPB1*894、DPB1*895、DPB1*896、DPB1*897、DPB1*898、DPB1*899、DPB1*90、DPB1*900、DPB1*901、DPB1*902、DPB1*903、DPB1*904、DPB1*905、DPB1*906、DPB1*907、DPB1*908、DPB1*909、DPB1*91、DPB1*910、DPB1*911、DPB1*912、DPB1*913、DPB1*914、DPB1*915、DPB1*916、DPB1*917、DPB1*918、DPB1*919、DPB1*92、DPB1*920、DPB1*921、DPB1*922, DPB1*923, DPB1*924, DPB1*925, DPB1*926, DPB1*927, DPB1*928, DPB1*929, DPB1*93, DPB1*930, DPB1*931, DPB1*932, DPB1*933 , DPB1*934, DPB1*935, DPB1*936, DPB1*937, DPB1*938, DPB1*939, DPB1*94, DPB1*940, DPB1*941, DPB1*942, DPB1*943, DPB1*944, DPB1*94 5, DPB1*946, DPB1*947, DPB1*948, DPB1*949, DPB1*95, DPB1*950, DPB1*951, DPB1*952, DPB1*953, DPB1*954, DPB1*955, DPB1*956, DPB1*957, DPB1*958, DPB1*959, DPB1*96, DPB1*960, DPB1*961, DPB1*962, DPB1*963, DPB1*964, DPB1*965, DPB1*97, DPB1*98, and DPB1*99.

[0021] In some embodiments, the DP beta chain comprises the amino acid sequence set forth in SEQ ID NO:1.

[0022] In some embodiments, the HLA class II molecule further comprises a DP alpha chain. In some embodiments, the DP alpha chain is selected from the group consisting of 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*01:03:0 1:20, 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*01:0 8, DPA1*01:09, DPA1*01:10, DPA1*01:11, DPA1*01:12, DPA1*01:13, DPA1*01:14, DPA1*01:15, DPA1*01:16, DPA1*01:17, DPA1*01:18, DPA1*01 :19, DPA1*02:01:01:01, DPA1*02:01:01:02, DPA1*02:01:01:03, DPA1*02:01:01:04, DPA1*02:01:01:05, DPA1*02:01:01:06, DPA1*02:01:01 :07, DPA1*02:01:01:08, DPA1*02:01:01:09, DPA1*02:01:01:10, DPA1*02:01:01:11, DPA1*02:01:02:01, DPA1*02:01:02:02, DPA1*02:01:03,DPA1*02:01:04, DPA1*02:01:05, DPA1*02:01:06, DPA1*02:01:07, DPA1*02:01:08:01, DPA1* 02:01:08:02, DPA1*02:02:02:01, DPA1*02:02:02:02, DPA1*02:02:02:03, DPA1*02:02:02:04 , DPA1*02:02:02:05, DPA1*02:02:03, DPA1*02:02:04, DPA1*02:02:05, DPA1*02:02:06, DPA1 *02:03, DPA1*02:04, DPA1*02:05, DPA1*02:06, DPA1*02:07:01:01, DPA1*02:07:01:02, DPA1* 02:07:01:03, DPA1*02:08, DPA1*02:09, DPA1*02:10, DPA1*02:11, DPA1*02:12, DPA1*02:13N , DPA1*02:14, DPA1*02:15, DPA1*02:16, DPA1*03:01:01:01, DPA1*03:01:01:02, DPA1*03:01: 01:03, DPA1*03:01:01:04, DPA1*03:01:01:05, DPA1*03:01:02, DPA1*03:02, DPA1*03:03, DPA1*03:04, DPA1*04:01:01:01, DPA1*04:01:01:02, and DPA1*04:01:01:03, DPA1*04:02.

[0023] In some embodiments, the DP alpha chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 6 or 8. In some embodiments, the DP alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 6 or 8.

[0024] In some embodiments, the HLA class II molecule is a DP1, DP2, DP3, DP4, DP5, DP6, DP8, or DP9 allele.

[0025] In some embodiments, the DP beta chain has a higher affinity for the CD4 protein compared to a reference HLA class II molecule, wherein the reference HLA class II molecule comprises a DP beta chain comprising (i) a leucine at the amino acid residue corresponding to position 112 of SEQ ID NO:1, and / or (ii) a valine at the amino acid residue corresponding to position 141 of SEQ ID NO:1.

[0026] In some embodiments, the affinity 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, or at least about 1000 times higher.

[0027] In some embodiments, the DP beta chain is bound to the membrane of a cell. In some embodiments, the DP beta chain is not bound to the membrane of a cell. In some embodiments, the DP beta chain comprises the extracellular domain of the full-length DP alpha chain. In some embodiments, the DP beta chain does not comprise the transmembrane domain of the full-length DP beta chain.

[0028] In some embodiments, the DP alpha chain is bound to the membrane of a cell. In some embodiments, the DP alpha chain is not bound to the membrane of a cell. In some embodiments, the DP alpha chain comprises the extracellular domain of the full-length DP alpha chain. In some embodiments, the DP alpha chain does not comprise the transmembrane domain of the full-length DP alpha chain.

[0029] In some embodiments, the DP beta chains are linked or bound to inert particles. In some embodiments, the inert particles are beads. In some embodiments, the inert particles are nanoparticles. In some embodiments, the nanoparticles are selected from PEGylated iron oxide, chitosan, dextran, gelatin, alginate, liposomes, starch, branched polymers, carbon-based carriers, polylactic acid, poly(cyano)acrylate, polyethyleinemine, block copolymers, polycaprolactone, SPIONS, USPIONS, Cd / Zn-selenide, or silica nanoparticles. In some embodiments, the nanoparticles are PEGylated iron oxide nanoparticles.

[0030] In some embodiments, the DP beta chain comprises a signal peptide. In some embodiments, the DP alpha chain comprises a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:9.

[0031] Certain aspects of the present disclosure relate to nucleic acid molecules encoding a DP beta chain as disclosed herein. In some embodiments, the nucleic acid molecule further encodes a DP alpha chain as disclosed herein.

[0032] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:2.

[0033] Certain aspects of the present disclosure pertain to vectors comprising the nucleic acid molecules disclosed herein.

[0034] Certain embodiments of the present disclosure relate to cells comprising the HLA class II molecules disclosed herein, the nucleic acid molecules disclosed herein, or the vectors disclosed herein. In some embodiments, the cells are mammalian cells or insect cells. In some embodiments, the cells are selected from K562 cells, T2, HEK293, HEK293T, A375, SK-MEL-28, Me275, COS, fibroblasts, tumor cells, or any combination thereof.

[0035] In some embodiments, the cells lack endogenous MHC class II DP beta chain expression. In some embodiments, the cells lack endogenous MHC class II DP alpha chain expression.

[0036] Certain aspects of the present disclosure relate to methods for identifying T cell receptors capable of binding to an epitope of an MHC class II complex, the methods comprising pulsing a cell as disclosed herein with one or more peptides comprising the epitope and detecting one or more CD4 + and stimulating the T cells with APCs.

[0037] Certain aspects of the present disclosure relate to methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an MHC class II molecule disclosed herein. In some embodiments, the disease or condition is cancer or an infection.

[0038] In some embodiments, the cancer is selected from the group consisting of melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, uterine cancer, lung cancer, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), esophageal cancer, small intestine cancer, urethral cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), bladder cancer, kidney or ureter cancer, renal pelvis cancer, glioma, squamous cell carcinoma, and combinations of the above cancers.

[0039] In some embodiments, the cancer is relapsed or refractory. In some embodiments, the cancer is locally advanced. In some embodiments, the cancer is progressive. In some embodiments, the cancer is metastatic.

[0040] In some embodiments, the HLA class II molecule has a K of less than about 100 μM. D In some embodiments, the HLA class II molecule binds to CD4 with a K of less than about 10 μM. D In some embodiments, the HLA class II molecule binds to CD4 with a K of about 8.9 μM or less. D It binds to CD4.

[0041] Certain aspects of the present disclosure relate to complexes comprising an HLA class II molecule disclosed herein and a peptide, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-237. [Brief explanation of the drawings]

[0042] [Figure 1-1](A)-(V) are graphical representations of data demonstrating that the affinity-matured DP4L112W / V141M molecule exhibits enhanced CD4 binding capacity. (A)-(F) are histograms showing the results of staining with anti-class II mAb and soluble CD4 (sCD4) on HLA class II null K562 cells stably expressing the wild-type DP α chain (DPA1*01:03) transduced with blank, wild-type, or mutant DP β chain (DPB1*04:01) with L112W, V114M, V141M, and M158I substitutions (DP4L112W / V114M / V141M / M158I). (G) is a graphical representation of data demonstrating that the affinity-matured DP4L112W / V141M molecule exhibits enhanced CD4 binding capacity. 1A-1F , and stained with sCD4. Figure 1B is a bar graph summarizing the binding affinity to sCD4 (MFI; Y axis) of all possible DP4 revertants expressed as in Figures 1A-1F and stained with sCD4. Figure 1H is a graphical representation of data showing that affinity-matured DP4L112W / V141M molecules exhibit enhanced CD4-binding capacity. Figure 1B shows the affinity between DP4L112W / V141M and CD4, as quantified by steady-state analysis. Figure 1C is a graphical representation of data showing that affinity-matured DP4L112W / V141M molecules exhibit enhanced CD4-binding capacity. Figures J-W show the results of IL-2 ELISPOT assays of DP4 / WT1 TCR-clone 9-transduced Jurkat76 and Jurkat76 / CD4 cells stimulated with wild-type DP4-expressing aAPCs or DP4L112W / V141M-expressing aAPCs pulsed with gradient concentrations of the DP4 / WT1 peptide. Graphical representations of data show that affinity-matured DP4L112W / V141M molecules exhibit enhanced CD4 binding capacity. Histograms show staining of K562 cells expressing the DP4L112W / V141M allele (as indicated) with anti-class II mAb and sCD4. Open histograms represent substandard control staining. *, P value less than 0.05 by Student's t-test. Bars and error bars represent the mean ± SD of triplicate experiments. At least two independent experiments were performed.X-AA are histograms showing wild-type DP4 and DP4L112W / V141M molecules on the surface of K562 cells, detected using the indicated anti-HLA class II antibodies. Staining of control cells lacking class II expression is shown in gray. AB-BH are histograms showing aAPCs expressing the indicated DP4s or parental class II cells stained with the indicated concentrations of sCD4. BI shows quantification of aAPCs expressing wild-type DP4 or DP4L112W / V141M at the indicated concentrations. Error bars represent the mean ± standard deviation of triplicate experiments. Experiments in Figures 1B-J and 1B-I were performed in parallel. B-J are biolayer interferometry sensorgrams showing the interaction of biotinylated wild-type DP4 (ligand) with sCD4 (analyte) over a range of concentrations. BK is a biolayer interferometry sensorgram showing the interaction of biotinylated DP4L112W / V141M (ligand) with sCD4 (analyte) over a range of concentrations. Experiments BJ and BI were performed in parallel. All data are representative of two independent experiments. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 2] Figures A–D are ribbon diagrams of the model structure of the DP4L112W / V141M and human CD4 complex. Figures A–B show two configurations of the model structure of the ternary complex of DPA1*01:03, DPB1*04:01, and CD4 as shown. The area enclosed by the dashed box is the DPB1*04:01-CD4 binding interface (B). Figures C–D are ribbon diagrams of the model structure of the DP4L112W / V141M and human CD4 complex. Zoomed-in views of the CD4-binding interface of wild-type DP4 (C) and DP4L112W / V141M (D) are shown. The side chains of interacting residues are shown in ball-and-stick representation (C–D). [Figure 3](A-P) Graphical representation of data showing staining of cognate TCRs expressed on human primary CD4+ T cells with DP4L112W / V141M dimers. Primary T cells were transduced with DP4 / MAGE-A3243-258 (R12C9; E-H), DP4 / WT1328-348 (clone 9; I-L), or DP4 / NY-ESO-1157-170 (5B8; M-P) TCRs and stained with the indicated DP4L112W / V141M dimers (B-D, F-H, J-L, and N-P). [Figure 4] (A-D) Scatter plots showing co-staining of R12C9 transduced CD4+ T cells stained with DP4L112W / V141M dimer and anti-Vβ22 mAb. Note that R12C9 expresses Vβ22. (E-H) Scatter plots showing co-staining of clone 9 transduced CD4+ T cells double-stained with DP4L112W / V141M dimer and anti-NGFR mAb. Note that the clone 9 and ΔNGFR genes are fused by P2A. [Figure 5] A–P are scatter plots showing co-staining of clone 9-transduced primary T cells (A–H) and 5B8-transduced primary T cells (I–P) stained with 5 μg / ml of conventional wild-type DP4 tetramer and DP4L112W / V141M dimer. At least two independent experiments were performed. [Figure 6-1] A–F are bar graphs showing the results of an extensive screening using the DP4L112W / V141M dimer, which recognized a series of novel DP4-restricted tumor-associated antigens. Peripheral CD4+ T cells were purified from six DP4+ melanoma patients, stimulated with DP4-expressing aAPCs individually pulsed with 196 different peptides derived from tumor-associated antigens, and stained with the cognate DP4L112W / V141M dimer. Results using the 30 most positive peptides are shown in A–B. Results for the remaining 166 peptides are shown in C–F. Each gating was set to show less than 0.2% positive control dimer staining. Positive dimer staining was defined as staining 3 standard deviations above the control dimer staining (>0.6%), as indicated by the dashed line. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 7-1] A–L are graphical representations of DP4L112W / V141M dimer staining of peptide-specific CD4+ T cells from melanoma patients. Primary CD4+ T cells were purified from six DP4+ melanoma patients, stimulated with DP4-expressing aAPCs individually pulsed with 196 different peptides derived from tumor-associated antigens, and stained with the cognate DP4L112W / V141M dimers indicated in A–F. Examples of DP4L112W / V141M dimer staining are shown. *: P value less than 0.05 by Student's t-test. ns: not significant. At least two independent experiments were performed. [Figure 7-2] Same as above. [Figure 8-1] AX are graphical representations of data showing that DP4-restricted TCRs isolated from DP4L112W / V141M dimer-positive cells and reconstituted with human TCR-deficient CD4+ T cells function in a DP4-restricted and antigen-specific manner. 03-CCND1219-238 (A–D), 05-HSD17B12225-244 and 09-HSD17B12225-244 (E–J), 05-LGSN296-315 (K–N), 03-MAGE-A2108-127 and 06-MAGE-A2108-127 (OT), and 05-MUC5AC4922-4941 (U–X) were cloned from DP4L112W / V141M dimer-positive cells, reconstituted in TCR-deficient Jurkat76 / CD4 cells, and stained with the corresponding DP4L112W / V141M dimer. [Figure 8-2] Same as above. [Figure 9]A-G are bar graphs showing the results of IL-2 ELISPOT assays of 03-CCND1219-238 (A), 05-HSD17B12225-244 (B), 09-HSD17B12225-244 (C), 05-LGSN296-315 (D), 03-MAGE-A2108-127 (E), 06-MAGE-A2108-127 (F), and 05-MUC5AC4922-4941 (G) stimulated with aAPCs pulsed with the corresponding peptides. DP4 / WT1 (clone 9) TCR was used as a negative control. At least two independent experiments were performed. *: P value less than 0.05 by Student's t-test. Bars and error bars represent the mean ± SD of triplicate experiments. [Figure 10-1] Figures A-Q are graphical representations of data demonstrating that DP4-restricted TCRs isolated from DP4L112W / V141M dimer-positive cells and reconstituted with human primary CD4+ T cells function in a DP4-restricted and antigen-specific manner. 03-CCND1219-238 (A-D and O), 03-MAGE-A2108-127 and 06-MAGE-A2108-127 (E-J and P), and 05-MUC5AC4922-4941 (K-N and Q) were retrovirally transduced into human primary CD4+ T cells and stained with the corresponding DP4L112W / V141M dimer (A-N). *: P value less than 0.05 by Student's t-test. ns: not significant. At least two independent experiments were performed. *: P value less than 0.05 by Student's t-test. Bars and error bars represent the mean ± SD of the results of three experiments. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 11]Figures 8A-8B show data demonstrating that DP4-restricted TCRs cloned from melanoma patients recognized peptides endogenously processed and presented by K562-based aAPCs. Figures 8A-8C show gel chromatography images showing CCDN1 (A) and MAGE-A2 (B) endogenously expressed in K562-derived aAPCs. Figures 8C-8D show data demonstrating that DP4-restricted TCRs cloned from melanoma patients recognized peptides endogenously processed and presented by K562-based aAPCs. Figures 8A-8C show bar graphs showing the results of IFN-γ ELISPOT assays of human primary T cells retrovirally transduced with 03-CCND1219-238 (C) or 06-MAGE-A2108-127 (D) and stimulated with non-peptide-pulsed HLA-null or DP4 aAPCs (C-D). (E) Data demonstrating that DP4-restricted TCRs cloned from melanoma patients recognized peptides endogenously processed and presented by K562-based aAPCs. (E) Bar graph showing the results of IFN-γ ELISPOT assays of human primary T cells retrovirally transduced with 05-MUC5AC4922-4941 TCRs and stimulated with MUC5AC4914-4949 minigene-transduced and non-peptide-pulsed HLA-null or DP4 aAPCs. At least two independent experiments were performed. *: P value less than 0.05 by Student's t-test. Bars and error bars represent the mean ± SD of triplicate experiments. [Figure 12]Figures A–E show data demonstrating that 06-MAGE-A2108-127 TCR recognizes melanoma cell lines in a DP4- and MAGE-A2-dependent manner. Figure A shows a Western blot image showing endogenous MAGE-A2 expression in K562 cells and the indicated melanoma cell lines. Figures B–E show bar graphs showing data from IFN-γ ELISPOT assays of primary human T cells transduced with 06-MAGE-A2108-127 TCR and stimulated with DP4-transduced SK-MEL-21 (DP4+MAGE-A2-; B) or SK-MEL-37 (DP4+MAGE-A2+; C), as well as SK-MEL-28 (DP4-MAGE-A2+; D) and Me275 (DP4-MAGE-A2+; E). *: P<0.05 by Student's t-test. Bars and error bars represent the mean ± SD of the results of three experiments. At least two independent experiments were performed. [Figure 13] A to Q 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 clones 9 to 49. Open histograms represent substandard control staining. [Figure 14] (A-D) Graphical representations of data showing the comparison of DP4L112W / V141M dimer and dextramer staining of endogenous TRPC1578-597-specific CD4 T cells. Endogenous (untransduced) TRPC1578-597-specific CD4 T cells were expanded from melanoma patients by stimulation with peptide-pulsed and irradiated DP4 artificial APCs and further stained with DP4L112W / V141M TRPC1578-597 dimer (B) or TRPC1578-597 dextramer (D). The corresponding CLIP multimers were used as controls (A and C). [Figure 15](A-F) Graphical representations of data showing the comparison of DP4L112W / V141M dimers with conventional DP4 tetramers and dextramers for staining of endogenous NY-ESO-1157-170-specific T cells. CD4+ T cells were purified from DP4+ healthy donor number 4 and stimulated once with NY-ESO-1157-170-pulsed and irradiated DP4+ artificial APCs. Expanded CD4+ T cells were individually stained with three different DP4 multimers (DP4L112W / V141M dimers (B), DP4 tetramers (D), or DP4 dextramers (F)) as indicated. [Figure 16] A-Y are graphical representations of data showing pathogen-specific CD4+ T cells subjected to ex vivo staining with DP4L112W / V141M dimers. Memory CD4+ T cells were purified from five DP4+ donors and subjected to ex vivo staining with DP4L112W / V141M dimers without in vitro stimulation against pathogen-associated peptides: TT948-968 (F-J), HSV-2-UL21283-302 (K-O), Flu-HA527-546 (P-T), and RSV-GP162-175 (U-Y). CLIP peptide was used as a negative control (A-E). [Figure 17-1](A-W) Graphical representations of data showing endogenous RSV-GP162-175-specific CD4+ T cell clones successfully constructed from DP4L112W / V141M dimer+ cells. Memory CD4+ T cells were purified from DP4+ donor #06 and subjected to ex vivo staining with DP4L112W / V141M RSV-GP162-175 dimers without in vitro stimulation. Dimer+ CD4+ T cells were then cloned by limiting dilution. (A-V) Graphical representations of representative dimer staining data for 10 dimer-positive and 1 dimer-negative single-cell clones. 77 of 84 clones (91.7%) successfully stained with DP4L112W / V141M RSV-GP162-175 dimers. W is a graphical representation of data showing the successful construction of endogenous RSV-GP162-175-specific CD4+ T cell clones from DP4L112W / V141M dimer+ cells. Memory CD4+ T cells were purified from DP4+ donor number 06 and subjected to ex vivo staining with DP4L112W / V141M RSV-GP162-175 dimers without in vitro stimulation. Dimer+ CD4+ T cells were then cloned by limiting dilution. This is a bar graph showing antigen-specific IL-2 production in RSV-GP162-175 dimer+ single cell clones. [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 18-1](A-S) Graphical representations of data showing endogenous DP4 TT948-968-specific CD4+ T cell clones successfully constructed from DP4L112W / V141M dimer+ cells. Memory CD4+ T cells were purified from DP4+ donor #04 and subjected to ex vivo staining with DP4L112W / V141M TTT948-968 dimers without in vitro stimulation. Dimer+ CD4+ T cells were then cloned by limiting dilution. (A-R) Graphical representations of representative dimer staining data for eight dimer-positive and one dimer-negative single-cell clones. Twenty-six of 29 clones (89.7%) successfully stained with DP4L112W / V141M TTT948-968 dimers. S is a graphical representation of data showing the successful construction of endogenous DP4 TT948-968-specific CD4+ T cell clones from DP4L112W / V141M dimer+ cells. Memory CD4+ T cells were purified from DP4+ donor number 04 and subjected to ex vivo staining with DP4L112W / V141M TT948-968 dimers without in vitro stimulation. Dimer+ CD4+ T cells were then cloned by limiting dilution. S is a bar graph showing antigen-specific IL-2 production in TT948-968 dimer+ single cell clones. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 19-1] A-NN are graphical representations of DP4 multimer staining of RSV-GP (Figures 19A-19P) and TT (Figures 19Q-19NN) dimer + single-cell clones. RSV-GP dimer + single-cell clones (c6, c12, c26, and c39) were stained with DP4L112W / V141M RSV-GP162-175 dimers (B, D, F, and H) or wild-type DP4 dextramers (J, L, N, and P). TT dimer+ single-cell clones (c2, c4, c6, and c9) were individually stained with three different DP4TT948-968 multimers: DP4L112W / V141M dimers (R, T, V, and X), wild-type DP4 tetramers (Z, BB, DD, and FF), and wild-type DP4 dextramers (HH, JJ, LL, and NN)). [Figure 19-2] Same as above. [Figure 19-3] Same as above. [Figure 19-4] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present disclosure relates to MHC class II molecules with enhanced affinity for CD4. In some embodiments, the present disclosure relates to MHC class II molecules comprising an HLA-DP (DP) beta chain, wherein the DP beta chain has enhanced affinity for CD4.

[0044] The present disclosure further relates to an MHC class II molecule comprising a DP beta chain, wherein the DP beta chain comprises an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1. In some embodiments, the DP beta chain further comprises an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1.

[0045] The present disclosure further relates to an MHC class II molecule comprising a DP beta chain, wherein the DP beta chain comprises an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1. In some embodiments, the DP beta chain further comprises an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1.

[0046] I. Terminology In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise stated herein, each of the following terms shall have the meaning indicated below. Additional definitions are set forth throughout this application.

[0047] It should be noted that the term "a" or "an" entity refers to one or more of that entity, for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0048] Furthermore, when used herein, "and / or" shall be construed as a specific disclosure of each of two particular features or components, with or without the other. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0049] The term "about" is used herein to mean approximately, roughly, roughly, or within a range. When the term "about" is used in conjunction with a numerical range, the term modifies that range by extending its boundaries above and below the set forth numerical values. In general, the term "about" is used herein to modify a numerical value by plus or minus 10 percent (above or below) the set forth value.

[0050] Whenever an embodiment is described herein using the word "comprising," it should be understood that other similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0051] 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 pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0052] Units, prefixes, and symbols are shown in their International System of Units (SI) accepted format. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not intended to limit the various aspects of the disclosure, which can be had by reference to the specification in its entirety. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0053] "Administering" refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Examples of routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes, for example, by injection or infusion. The phrase "parenteral administration," as used herein, refers to a method of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, for example, orally. Other non-parenteral routes include topical, epithelial, or mucosal administration routes, such as intranasally, intravaginally, rectally, sublingually, or topically, and administration can be, for example, single, multiple, and / or over one or more extended periods of time.

[0054] The term "HLA" as used herein refers to human leukocyte antigens. HLA genes encode human major histocompatibility complex (MHC) proteins. MHC proteins are expressed on the surface of cells and are involved in activating immune responses. HLA class II genes encode MHC class II proteins expressed on the surface of professional antigen-presenting cells (APCs). Non-limiting examples of professional APCs include monocytes, macrophages, dendritic cells (DCs), and B lymphocytes. Some endothelial and epithelial cells can also express MHC class II molecules after inflammatory signals are activated. Humans lacking functional MHC class II molecules are highly susceptible to a range of infectious diseases and usually die at an early age.

[0055] 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. Thus, "HLA class II molecule" and "MHC class II molecule" are used interchangeably herein. A typical MHC class II molecule comprises two protein chains, an alpha chain and a beta chain. Generally, each of the naturally occurring alpha and beta chains contains a transmembrane domain that anchors the alpha / beta chain to the cell surface and an extracellular domain that carries antigen and interacts with TCR and / or CD4 expressed on T cells.

[0056] Both the alpha and beta chains of MHC class II are encoded by the HLA gene complex. The HLA complex is located in the 6p21.3 region of the short arm of human chromosome 6 and contains more than 220 genes of diverse functions. The HLA gene complex contains many variants with more than 20,000 HLA alleles and related alleles, including more than 250 MHC class II alpha chain alleles and more than 5,000 MHC class II beta chain alleles, encoding thousands of MHC class II proteins, as known in the art (see, e.g., hla.alleles.org, last visited May 20, 2019, incorporated herein by reference in its entirety). For example, one such HLA-DP allele, DP4, is the most frequently found allele in many ethnic groups. Each alpha and beta chain is usually expressed as a proprotein, which further contains a cleaved signal peptide. Any number of naturally occurring signal peptides may be used to facilitate expression and localization of the alpha and beta chains disclosed herein. One such example is SEQ ID NO:9.

[0057] 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 organization and function. Representative HLA-DP sequences are shown in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0058] When MHC class II molecules form a complex with an antigenic peptide, the 10-30 amino acid-long antigenic peptide binds to the peptide-binding groove and is presented extracellularly to CD4+ cells. Both the alpha and beta chains fold into two separate domains: alpha 1 and alpha 2 for the alpha polypeptide and beta 1 and beta 2 for the beta polypeptide. The constant residues L112, V114, V141, L156, and M158, which are recognized and bound by CD4, are located in the beta 2 domain of the beta polypeptide. The open peptide-binding groove, which holds the antigen to be presented, is located between the alpha 1 and beta 1 domains. Upon interaction with CD4+ T cells, the MHC class II complex interacts with the T cell receptor (TCR) expressed on the surface of the T cell. In addition, the beta chain of the MHC class II molecule interacts only slightly with CD4 expressed on the surface of the T cell (K). D >2mM). The standard CD4 amino acid sequence (SEQ ID NO: 10) is shown in Table 2 (UniProt - P01730). [Table 2]

[0059] As used herein, the term "T cell receptor" (TCR) refers to a heteromeric cell surface receptor that can specifically interact with a target antigen. As used herein, the term "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 are responsible for T cell recognition and targeting of antigen-presenting cells. Antigen-presenting cells (APCs) present fragments of foreign proteins (antigens) complexed with major histocompatibility complexes (MHC class I or MHC class II, also referred to herein as those complexed with HLA molecules, e.g., HLA class II molecules). The TCR recognizes and binds to the peptide:HLA complex, recruiting CD8 (in the case of MHC class I molecules) or CD4 (in the case of MHC class II molecules) expressed on the T cell, thereby activating the TCR. The activated TCR initiates downstream signaling and immune responses, including the destruction of the APC.

[0060] Generally, a TCR may comprise two chains, an alpha chain and a beta chain (or less commonly, a gamma chain and a delta chain), interconnected by disulfide bonds. Each chain comprises a variable domain (an alpha chain variable domain and a beta chain variable domain) and a constant region (an alpha chain constant region and a beta chain constant region). The variable domains are located distal to the cell membrane, and the variable domains interact with antigen. The constant regions are located proximal to the cell membrane. A TCR may further comprise a transmembrane region and a short cytoplasmic tail. As used herein, the term "constant region" encompasses the transmembrane region and cytoplasmic tail, if present, as well as the conventional "constant region."

[0061] Variable domains can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), flanked by more conserved regions called framework regions (FRs). Each alpha and beta 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 an antigen. Although all three CDRs of each chain are involved in antigen binding, CDR3 is considered to be the primary antigen-binding region, and CDR1 and CDR2 are thought to primarily recognize HLA molecules.

[0062] Unless explicitly stated, and unless the context dictates otherwise, the term "TCR" also includes antigen-binding fragments or portions of any TCR disclosed herein, including monovalent and bivalent fragments or portions, and single-chain TCRs. The term "TCR" is not limited to naturally occurring TCRs bound to the surface of T cells. As used herein, the term "TCR" also refers to a TCR described herein expressed on the surface of a cell other than a T cell (e.g., a cell that naturally expresses or is modified to express CD4 as described herein), or a TCR described herein free from the cell membrane (e.g., an isolated TCR or a soluble TCR).

[0063] "Antigen-binding molecule," "portion of a TCR," or "TCR fragment" refers to any portion of a TCR that is smaller than the entire TCR. An antigen-binding molecule may include the CDRs of an antigen.

[0064] "Antigen" refers to any molecule, e.g., a peptide, that can elicit an immune response or be bound by a TCR. As used herein, "epitope" refers to a portion of a polypeptide that can elicit an immune response or be bound by a TCR. The immune response may include antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will readily understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Antigens and / or epitopes can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed. Antigens and / or epitopes can be specific to a particular tissue, such as cancer cells, or they can be broadly expressed. Furthermore, fragments of larger molecules can function as antigens. In one embodiment, the antigen is a tumor antigen. An epitope can be present in a longer polypeptide (e.g., in a protein), or it can exist as a fragment of a longer polypeptide. In some embodiments, the epitope forms a complex with a major histocompatibility complex (MHC, also referred to herein as complexing with an HLA molecule, eg, an HLA class I molecule).

[0065] The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced. For example, autologous T cell therapy involves administering T cells isolated from the same subject to a subject. The term "allogeneic" refers to any material derived from one individual that is then 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.

[0066] "Cancer" refers to a broad group of diseases characterized by the unchecked growth of abnormal cells in the body. Uncontrolled cell division and growth leads to the formation of malignant tumors that infiltrate neighboring tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous 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 lymphomas, leukemias, and other white blood cell malignancies. In some embodiments, the methods of the present invention can be used to reduce the size of tumors derived from, for example, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, uterine cancer, lung cancer, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), esophageal cancer, small intestine cancer, urethral cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), bladder cancer, kidney or ureter cancer, renal pelvis cancer, glioma, squamous cell carcinoma, and combinations of the above cancers. Certain cancers may respond to chemotherapy or radiation therapy, or the cancer may be refractory. Refractory cancer refers to cancer that cannot be corrected by surgical intervention, and the cancer does not respond to chemotherapy or radiation therapy from the beginning or becomes unresponsive over time.

[0067] As used herein, the term "progression-free survival", which may be abbreviated as PFS, 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 response criteria for malignant lymphoma.

[0068] The term "overall survival," sometimes abbreviated as OS, is defined as the time from the date of treatment to the date of death.

[0069] As used herein, the term "infection" refers to some type of invasion of one or more tissues of the body by a foreign substance. The term "infection" includes, but is not limited to, infection by viruses (including viroids and prions), bacteria, fungi, parasites, and any combination thereof.

[0070] The term "lymphocyte" as used herein includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cytotoxic) lymphocyte and a major component of the innate immune system. NK cells eliminate tumor- and virus-infected cells. They function through the process of apoptosis, or programmed cell death. They were called "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without the involvement of antibodies). T cell receptors (TCRs) distinguish T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is 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 (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem memory T cells such as naive cells), and (ii) naive T cells. SCM The cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and exhibit numerous functional attributes characteristic of memory cells. (ii) Central memory T CM The cells express L-selectin and CCR7, they secrete IL-2 but not IFNγ or IL-4, and (iii) effector memory T EM These include T cells (which do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma-delta T cells. B cells, on the other hand, play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, hence their name.

[0071] As used herein with respect to nucleotide or amino acid sequences, the terms "modified" and "mutated" refer to a change in the sequence compared to a wild-type sequence or a specific reference sequence. The terms "modified" and "mutated," unless otherwise specified, do not require a process step to create the modified or mutated sequence (e.g., a modified beta chain sequence). Rather, these terms indicate that there is a change in the modified or mutated sequence compared to a reference sequence, e.g., a wild-type sequence. For example, a DP beta chain containing a substitution mutation at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 does not require that the wild-type DP beta chain be physically altered to arrive at the recited DP beta chain, but rather, when properly sequenced, the recited DP beta chain contains an amino acid residue at the recited position (residue 112) that differs from the amino acid residue at the corresponding position in the wild-type or reference DP beta chain.

[0072] 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 (-NH) functional group, (ii) a carboxyl (-COOH) functional group, and (iii) a side chain (R group), where the side chain is unique 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, 20 of which are encoded by the genetic code. Amino acids with positively charged side chains include arginine (Arg; R), histidine (His, H), and lysine (Lys; K). Amino acids with 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) may also be classified as polar and / or amphipathic, in that these amino acids are often found on the surface of proteins or lipid membranes. Additional amino acids include cysteine ​​(Cys; C), selenocysteine ​​(Sec; U), glycine (Gly; G), and proline (Pro; P).

[0073] As used herein, "corresponding to position" is used as a means of identifying a specific amino acid residue, for example, a specific amino acid position in a polynucleotide, or a specific nucleic acid, for example, a specific nucleic acid position in a polypeptide. The position can be determined by appropriately aligning the sequence of interest 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 visited: May 25, 2019).

[0074] The terms "genetically modified" or "engineered" refer to methods of modifying the genome of a cell, including, but not limited to, deleting coding or non-coding regions or portions thereof, or inserting coding regions or portions thereof. In some embodiments, the modified cells are lymphocytes, e.g., T cells, or modified cells expressing CD4, which can be obtained from a patient or a donor. The cells can be modified to express an exogenous construct, such as a T cell receptor (TCR) disclosed herein, that is integrated into the genome of the cell. In some embodiments, the cells are modified to express CD4.

[0075] "Immune response" refers to the actions 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 antibodies (Abs), cytokines, and complement) produced by any of these cells or the liver that result in the elimination of invading pathogens from the vertebrate body and / or the selective targeting, binding, damage, and destruction of pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0076] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from or recurring with a disease by methods involving inducing, enhancing, suppressing, or otherwise modifying the immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy includes adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation.

[0077] Cells used in the immunotherapies described herein can be derived from sources known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be obtained from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood drawn from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Additional 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 can also include administering modified cells to a subject, wherein the modified cells express CD4 and TCR as disclosed herein. In some embodiments, the modified cells are not T cells.

[0078] As used herein, a "patient" includes any human suffering from cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0079] 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 a protein or peptide sequence may contain. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, as well as longer chains, of which there are many varieties, commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. The polypeptide may be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0080] As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, mediating a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, e.g., a T cell receptor (TCR) / CD4 complex, that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (e.g., an aAPC, a dendritic cell, a B cell, etc.), specifically binds to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include, but are not limited to, MHC class II molecules bearing peptides, anti-CD4 antibodies, anti-CD28 antibodies, anti-CD2 antibodies, and anti-CD3 antibodies.

[0081] "Treatment" or "treating" a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, symptom severity, or recurrence of a symptom, complication, or condition, or biochemical manifestations associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission.

[0082] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of the listed or shown components.

[0083] The terms "about" or "essentially comprising" refer to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" can mean within one standard deviation or more than one standard deviation, per practice in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 10% (i.e., ±10%). For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (for 10%). Furthermore, particularly with respect to biological systems or processes, the term can mean a value up to an order of magnitude or up to five times greater. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range of that particular value or composition.

[0084] As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0085] Various aspects of the invention are described in further detail in the following subsections.

[0086] II. Compositions of the Present Disclosure The present disclosure relates to HLA class II molecules with enhanced CD4 binding. Certain embodiments of the present disclosure relate to HLA class II molecules comprising a beta chain, wherein the beta chain comprises one or more mutations. In certain embodiments, the one or more mutations in the beta chain enhance the affinity of the beta chain for CD4. In certain embodiments, the beta chain is an HLA-DP ("DP") beta chain. II.A. MHC class II molecules

[0087] The human leukocyte antigen (HLA) system (human major histocompatibility complex [MHC]) is an important part of the immune system and is controlled by genes on chromosome 6. It encodes cell surface molecules specialized for presenting antigenic peptides to the T cell receptor (TCR) on T cells. (See Overview of the Immune System.) MHC molecules that present antigens (Ags) are divided into two major classes: class I MHC molecules and class II MHC molecules.

[0088] MHC class II molecules are present as transmembrane glycoproteins on the surface of professional antigen-presenting cells (APCs). Intact class II molecules are composed of an alpha chain and a beta chain. Three loci in the HLA complex encode MHC class II proteins: HLA-DP, HLA-DQ, and HLA-DR. T cells expressing CD4 molecules react with MHC class II molecules. These lymphocytes often have effector and helper functions, activating responses to eliminate autologous cells infected with intracellular pathogens or destroy extracellular parasites, and providing support to other T cells, such as CD8 T cells. Because only professional APCs express MHC class II molecules, only these cells present antigens to CD4 T cells (CD4 binds to the non-polymorphic portions of the alpha2 and beta2 domains of the alpha and beta chains of MHC class II molecules, respectively).

[0089] In some embodiments, the HLA class II alpha and beta chains are selected from HLA-DP, HLA-DQ, and HLA-DR alleles. In certain embodiments, the HLA class II beta chain is an HLA-DP allele. In certain embodiments, the HLA class II alpha chain is an HLA-DP allele.

[0090] Many HLA-DP alleles are known in the art, and any known alleles can be used in the present disclosure. Examples of HLA-DP alpha and beta chain alleles are shown in Table 1. An updated list of HLA alleles is available at hla.alleles.org / (last visited February 27, 2019).

[0091] II.A.1. MHC class II beta chain In certain embodiments, the HLA class II molecule comprises a DP beta chain, wherein the DP beta chain comprises an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1. Any amino acid other than leucine may be present at the amino acid residue corresponding to position 112 of SEQ ID NO: 1. In some embodiments, the amino acid other than leucine is an amino acid comprising a hydrophobic side chain. In certain embodiments, the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 is an amino acid selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain embodiments, the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 is alanine. In certain embodiments, the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 is valine. In certain embodiments, the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 is isoleucine. In certain embodiments, the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 is methionine. In certain embodiments, the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 is phenylalanine. In certain embodiments, the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 is tyrosine. In certain embodiments, the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 is tryptophan.

[0092] In some embodiments, the non-leucine amino acid at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 is made up of two or more amino acids, e.g., two amino acids, three amino acids, four amino acids, five amino acids, or more. In some embodiments, at least one of the two or more amino acids comprises a hydrophobic side chain. In certain embodiments, the non-leucine amino acid at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 is made up of a series of amino acids, e.g., at least two, at least three, at least four, or at least five, where each amino acid in the series comprises a hydrophobic side chain.

[0093] In certain embodiments, the HLA class II molecule comprises a DP beta chain, wherein the DP beta chain comprises an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1. Any amino acid other than valine may be present at the amino acid residue corresponding to position 141 of SEQ ID NO: 1. In some embodiments, the amino acid other than valine is an amino acid comprising a hydrophobic side chain. In certain embodiments, the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1 is an amino acid selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain embodiments, the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1 is alanine. In certain embodiments, the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1 is isoleucine. In certain embodiments, the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1 is leucine. In certain embodiments, the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1 is methionine. In certain embodiments, the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1 is phenylalanine. In certain embodiments, the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1 is tyrosine. In certain embodiments, the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1 is tryptophan.

[0094] In some embodiments, the non-valine amino acid residue at position 141 of SEQ ID NO: 1 is made up of two or more amino acids, e.g., two, three, four, five, or more amino acids. In some embodiments, at least one of the two or more amino acids comprises a hydrophobic side chain. In certain embodiments, the non-valine amino acid residue at position 141 of SEQ ID NO: 1 is made up of a series of amino acids, e.g., at least two, at least three, at least four, or at least five, where each amino acid in the series comprises a hydrophobic side chain.

[0095] In certain embodiments of the present disclosure, the MHC class II molecule comprises a DP beta chain that comprises two or more substitution mutations compared to the wild-type DP beta chain.In certain embodiments, the DP beta chain comprises at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, at least eight mutations, at least nine mutations, or at least ten mutations compared to the wild-type DP beta chain.

[0096] In certain embodiments, the DP beta chain comprises an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 and an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1. In some embodiments, (i) the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1, (ii) the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1, or each of the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 and the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1 is an amino acid comprising a hydrophobic side chain. In some embodiments, (i) the amino acid other than leucine at the amino acid residue corresponding to position 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 amino acid residue corresponding to position 141 of SEQ ID NO: 1 is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0097] In some embodiments, (i) the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO:1 is tryptophan; and (ii) the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO:1 is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some embodiments, (i) the amino acid other than leucine at the amino acid residue corresponding to position 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 amino acid residue corresponding to position 141 of SEQ ID NO:1 is methionine. In some embodiments, (i) the amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO:1 is tryptophan; and (ii) the amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO:1 is methionine.

[0098] In certain embodiments, the DP beta chain further comprises an amino acid other than valine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1. In some embodiments, the amino acid other than valine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1 is selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain embodiments, the amino acid other than valine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1 is methionine.

[0099] In certain embodiments, the DP beta chain further comprises an amino acid other than methionine at the amino acid residue corresponding to position 158 of SEQ ID NO: 1. In some embodiments, the amino acid other than methionine at the amino acid residue corresponding to position 158 of SEQ ID NO: 1 is selected from alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In certain embodiments, the amino acid other than methionine at the amino acid residue corresponding to position 158 of SEQ ID NO: 1 is isoleucine.

[0100] In some embodiments, the DP beta chain comprises (i) an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1, and (ii) an amino acid other than valine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1. In some embodiments, the DP beta chain comprises (i) an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1, and (ii) an amino acid other than methionine at the amino acid residue corresponding to position 158 of SEQ ID NO: 1.

[0101] In some embodiments, the DP beta chain comprises (i) a tryptophan at the amino acid residue corresponding to position 112 of SEQ ID NO: 1, and (ii) a methionine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1. In some embodiments, the DP beta chain comprises (i) a tryptophan at the amino acid residue corresponding to position 112 of SEQ ID NO: 1, and (ii) an isoleucine at the amino acid residue corresponding to position 158 of SEQ ID NO: 1.

[0102] In some embodiments, the DP beta chain comprises (i) an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO:1, and (ii) an amino acid other than valine at the amino acid residue corresponding to position 114 of SEQ ID NO:1. In some embodiments, the DP beta chain comprises (i) an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO:1, and (ii) an amino acid other than methionine at the amino acid residue corresponding to position 158 of SEQ ID NO:1.

[0103] In some embodiments, the DP beta chain comprises (i) a methionine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1, and (ii) an amino acid other than valine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1. In some embodiments, the DP beta chain comprises (i) a methionine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1, and (ii) an isoleucine at the amino acid residue corresponding to position 158 of SEQ ID NO: 1.

[0104] In some embodiments, the DP beta chain comprises (i) an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO:1, and (ii) an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO:1, (iii) an amino acid other than valine at the amino acid residue corresponding to position 114 of SEQ ID NO:1, and (iv) an amino acid other than methionine at the amino acid residue corresponding to position 158 of SEQ ID NO:1.

[0105] In some embodiments, the DP beta chain comprises (i) a tryptophan at the amino acid residue corresponding to position 112 of SEQ ID NO:1, (ii) a methionine at the amino acid residue corresponding to position 141 of SEQ ID NO:1, (iii) a methionine at the amino acid residue corresponding to position 114 of SEQ ID NO:1, and (iv) an isoleucine at the amino acid residue corresponding to position 158 of SEQ ID NO:1.

[0106] In some embodiments, the DP beta chain comprises a valine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1. In some embodiments, the DP beta chain comprises a methionine at the amino acid residue corresponding to the corresponding position 158 of SEQ ID NO: 1. In some embodiments, the DP beta chain comprises (i) an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1, (ii) an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1, and (iii) a valine at the amino acid residue corresponding to position 114 of SEQ ID NO: 1. In some embodiments, the DP beta chain comprises (i) an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1, (ii) an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1, and (iii) a methionine at the amino acid residue corresponding to the corresponding position 158 of SEQ ID NO: 1. In some embodiments, the DP beta chain comprises (i) an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO:1, (ii) an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO:1, (iii) a valine at the amino acid residue corresponding to position 114 of SEQ ID NO:1, and (iv) a methionine at the amino acid residue corresponding to the corresponding position 158 of SEQ ID NO:1.

[0107] In some embodiments, the DP beta chain comprises (i) a tryptophan at the amino acid residue corresponding to position 112 of SEQ ID NO:1, (ii) a methionine at the amino acid residue corresponding to position 141 of SEQ ID NO:1, and (iii) a valine at the amino acid residue corresponding to position 114 of SEQ ID NO:1. In some embodiments, the DP beta chain comprises (i) a tryptophan at the amino acid residue corresponding to position 112 of SEQ ID NO:1, (ii) a methionine at the amino acid residue corresponding to position 141 of SEQ ID NO:1, and (iii) a methionine at the amino acid residue corresponding to the corresponding position 158 of SEQ ID NO:1. In some embodiments, the DP beta chain comprises (i) a tryptophan at the amino acid residue corresponding to position 112 of SEQ ID NO:1, (ii) a methionine at the amino acid residue corresponding to position 141 of SEQ ID NO:1, (iii) a valine at the amino acid residue corresponding to position 114 of SEQ ID NO:1, and (iv) a methionine at the amino acid residue corresponding to the corresponding position 158 of SEQ ID NO:1.

[0108] In some embodiments, the DP beta chain comprises (i) a tryptophan at the amino acid residue corresponding to position 112 of SEQ ID NO:1, (ii) a methionine at the amino acid residue corresponding to position 141 of SEQ ID NO:1, (iii) a methionine at the amino acid residue corresponding to position 114 of SEQ ID NO:1, and (iv) an isoleucine at the amino acid residue corresponding to position 158 of SEQ ID NO:1.

[0109] In certain embodiments, the DP beta chains described herein have a higher affinity for CD4 protein compared to a reference HLA class II molecule. In some embodiments, the reference HLA class II molecule is an HLA class II molecule having a wild-type DP beta chain. In some embodiments, the reference HLA class II molecule is an HLA class II molecule having a DP beta chain containing (i) a leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1, and / or (ii) a valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1.

[0110] In some embodiments, the enhanced affinity for CD4 is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 1000-fold, at least about 1500-fold, at least about 2000-fold, at least about 2500-fold, at least about 3000-fold, at least about 3500-fold, at least about 4000-fold, at least about 4500-fold, or at least about 4000-fold greater than the affinity of a reference HLA class II molecule for CD4.

[0111] In some embodiments, the enhanced affinity for CD4 is at least about 1.5 times to at least about 5000 times, 1.5 times to at least about 4000 times, 1.5 times to at least about 3000 times, 1.5 times to at least about 2000 times, 1.5 times to at least about 1000 times, 10 times to at least about 5000 times, 10 times to at least about 4000 times, 10 times to at least about 3000 times, 10 times to at least about 2000 times, 10 times to at least about 1000 times, 10 times to at least about 900 times, 10 times to at least about 800 times, 10 times to at least about 700 times, 10 times to at least about 600 times, 10 times at least about 500 times, 10 times to at least about 400 times, 10 times to at least about 300 times, 10 times to at least about 200 times, 10 times to at least about 100 times, 100 times to at least about 5000 times, 100 times to at least about 4000 times, 100 times to at least about 3000 times, 100 times to at least about 2000 times, 100 times to at least about 1000 times, 100 times to at least about 900 times, 100 times to at least about 800 times, 100 times to at least about 700 times, 100 times to at least about 600 times, 100 times to at least about 500 times, 100 times to at least about 400 times, 100 times to at least about 300 times, or 100 times to at least about 200 times higher.

[0112] In certain embodiments, the DP beta chain is 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*11, DPB1*110, DPB1*111, DPB1*112, DPB1*113, DPB1*114, DPB1*115, DPB1 *116, DPB1*117, DPB1*118, DPB1*119, DPB1*120, DPB1*121, DPB1*122, DPB1*123, DPB1*124, DPB1*125, DPB1*126, DPB1*127, DPB1*128, DPB1*129, DP B1*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, DP B1*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, D PB1*155, DPB1*156, DPB1*157, DPB1*158, DPB1*159, DPB1*16, DPB1*160, DPB1*161, DPB1*162, DPB1*163, DPB1*164, DPB1*165, DPB1*166, DPB1*167, DPB1*168, DPB1*169, DPB1*17, DPB1*170, DPB1*171, DPB1*172, DPB1*173, DPB1*174, DPB1*175, DPB1*176, DPB1*177, DPB1*178, DPB1*179, DPB1*18, DPB1*180, DPB1*181, DPB1*182, DPB1*183, DPB1*184, DPB1*185, DPB1*186, DPB1*187, DPB1*188, DPB1*189, DPB1*19, DPB1*190, DPB1*191, DPB1*192,DPB1*193、DPB1*194、DPB1*195、DPB1*196、DPB1*197、DPB1*198、DPB1*199、DPB1*20、DPB1*200、DPB1*201、DPB1*202、DPB1*203、DPB1*204、DPB1*205、DPB1*206、DPB1*207、DPB1*208、DPB1*209、DPB1*21、DPB1*210、DPB1*211、DPB1*212、DPB1*213、DPB1*214、DPB1*215、DPB1*216、DPB1*217、DPB1*218、DPB1*219、DPB1*22、DPB1*220、DPB1*221、DPB1*222、DPB1*223、DPB1*224、DPB1*225、DPB1*226、DPB1*227、DPB1*228、DPB1*229、DPB1*23、DPB1*230、DPB1*231、DPB1*232、DPB1*233、DPB1*234、DPB1*235、DPB1*236、DPB1*237、DPB1*238、DPB1*239、DPB1*24、DPB1*240、DPB1*241、DPB1*242、DPB1*243、DPB1*244、DPB1*245、DPB1*246、DPB1*247、DPB1*248、DPB1*249、DPB1*25、DPB1*250、DPB1*251、DPB1*252、DPB1*253、DPB1*254、DPB1*255、DPB1*256、DPB1*257、DPB1*258、DPB1*259、DPB1*26、DPB1*260、DPB1*261、DPB1*262、DPB1*263、DPB1*264、DPB1*265、DPB1*266、DPB1*267、DPB1*268、DPB1*269、DPB1*27、DPB1*270、DPB1*271、DPB1*272、DPB1*273、DPB1*274、DPB1*275、DPB1*276、DPB1*277、DPB1*278、DPB1*279、DPB1*28、DPB1*280、DPB1*281、DPB1*282、DPB1*283、DPB1*284、DPB1*285、DPB1*286、DPB1*287、DPB1*288、DPB1*289、DPB1*29、DPB1*290、DPB1*291、DPB1*292、DPB1*293、DPB1*294、DPB1*295、DPB1*296、DPB1*297、DPB1*298、DPB1*299、DPB1*30、DPB1*300、DPB1*301、DPB1*302、DPB1*303、DPB1*304、DPB1*305、DPB1*306、DPB1*307、DPB1*308、DPB1*309、DPB1*31、DPB1*310、DPB1*311、DPB1*312、DPB1*313、DPB1*314、DPB1*315、DPB1*316、DPB1*317、DPB1*318、DPB1*319、DPB1*32、DPB1*320、DPB1*321、DPB1*322、DPB1*323、DPB1*324、DPB1*325、DPB1*326、DPB1*327、DPB1*328、DPB1*329、DPB1*33、DPB1*330、DPB1*331、DPB1*332、DPB1*333、DPB1*334、DPB1*335、DPB1*336、DPB1*337、DPB1*338、DPB1*339、DPB1*34、DPB1*340、DPB1*341、DPB1*342、DPB1*343、DPB1*344、DPB1*345、DPB1*346、DPB1*347、DPB1*348、DPB1*349、DPB1*35、DPB1*350、DPB1*351、DPB1*352、DPB1*353、DPB1*354、DPB1*355、DPB1*356、DPB1*357、DPB1*358、DPB1*359、DPB1*36、DPB1*360、DPB1*361、DPB1*362、DPB1*363、DPB1*364、DPB1*365、DPB1*366、DPB1*367、DPB1*368、DPB1*369、DPB1*37、DPB1*370、DPB1*371、DPB1*372、DPB1*373、DPB1*374、DPB1*375、DPB1*376、DPB1*377、DPB1*378、DPB1*379、DPB1*38、DPB1*380、DPB1*381、DPB1*382、DPB1*383、DPB1*384、DPB1*385、DPB1*386、DPB1*387、DPB1*388、DPB1*389、DPB1*39、DPB1*390、DPB1*391、DPB1*392、DPB1*393、DPB1*394、DPB1*395、DPB1*396、DPB1*397、DPB1*398、DPB1*399、DPB1*40、DPB1*400、DPB1*401、DPB1*402、DPB1*403、DPB1*404、DPB1*405、DPB1*406、DPB1*407、DPB1*408、DPB1*409、DPB1*41、DPB1*410、DPB1*411、DPB1*412、DPB1*413、DPB1*414、DPB1*415、DPB1*416、DPB1*417、DPB1*418、DPB1*419、DPB1*420、DPB1*421、DPB1*422、DPB1*423、DPB1*424、DPB1*425、DPB1*426、DPB1*427、DPB1*428、DPB1*429、DPB1*430、DPB1*431、DPB1*432、DPB1*433、DPB1*434、DPB1*435、DPB1*436、DPB1*437、DPB1*438、DPB1*439、DPB1*44、DPB1*440、DPB1*441、DPB1*442、DPB1*443、DPB1*444、DPB1*445、DPB1*446、DPB1*447、DPB1*448、DPB1*449、DPB1*45、DPB1*450、DPB1*451、DPB1*452、DPB1*453、DPB1*454、DPB1*455、DPB1*456、DPB1*457、DPB1*458、DPB1*459、DPB1*46、DPB1*460、DPB1*461、DPB1*462、DPB1*463、DPB1*464、DPB1*465、DPB1*466、DPB1*467、DPB1*468、DPB1*469、DPB1*47、DPB1*470、DPB1*471、DPB1*472、DPB1*473、DPB1*474、DPB1*475、DPB1*476、DPB1*477、DPB1*478、DPB1*479、DPB1*48、DPB1*480、DPB1*481、DPB1*482、DPB1*483、DPB1*484、DPB1*485、DPB1*486、DPB1*487、DPB1*488、DPB1*489、DPB1*49、DPB1*490、DPB1*491、DPB1*492、DPB1*493、DPB1*494、DPB1*495、DPB1*496、DPB1*497、DPB1*498、DPB1*499、DPB1*50、DPB1*500、DPB1*501、DPB1*502、DPB1*503、DPB1*504、DPB1*505、DPB1*506、DPB1*507、DPB1*508、DPB1*509、DPB1*51、DPB1*510、DPB1*511、DPB1*512、DPB1*513、DPB1*514、DPB1*515、DPB1*516、DPB1*517、DPB1*518、DPB1*519、DPB1*52、DPB1*520、DPB1*521、DPB1*522、DPB1*523、DPB1*524、DPB1*525、DPB1*526、DPB1*527、DPB1*528、DPB1*529、DPB1*53、DPB1*530、DPB1*531、DPB1*532、DPB1*533、DPB1*534、DPB1*535、DPB1*536、DPB1*537、DPB1*538、DPB1*539、DPB1*54、DPB1*540、DPB1*541、DPB1*542、DPB1*543、DPB1*544、DPB1*545、DPB1*546、DPB1*547、DPB1*548、DPB1*549、DPB1*55、DPB1*550、DPB1*551、DPB1*552、DPB1*553、DPB1*554、DPB1*555、DPB1*556、DPB1*557、DPB1*558、DPB1*559、DPB1*56、DPB1*560、DPB1*561、DPB1*562、DPB1*563、DPB1*564、DPB1*565、DPB1*566、DPB1*567、DPB1*568、DPB1*569、DPB1*57、DPB1*570、DPB1*571、DPB1*572、DPB1*573、DPB1*574、DPB1*575、DPB1*576、DPB1*577、DPB1*578、DPB1*579、DPB1*58、DPB1*580、DPB1*581、DPB1*582、DPB1*583、DPB1*584、DPB1*585、DPB1*586、DPB1*587、DPB1*588、DPB1*589、DPB1*59、DPB1*590、DPB1*591、DPB1*592、DPB1*593、DPB1*594、DPB1*595、DPB1*596、DPB1*597、DPB1*598、DPB1*599、DPB1*60、DPB1*600、DPB1*601、DPB1*602、DPB1*603、DPB1*604、DPB1*605、DPB1*606、DPB1*607、DPB1*608、DPB1*609、DPB1*61、DPB1*610、DPB1*611、DPB1*612、DPB1*613、DPB1*614、DPB1*615、 DPB1*616、DPB1*617、DPB1*618、DPB1*619、DPB1*62、DPB1*620、DPB1*621、DPB1*622、DPB1*623、DPB1*624、DPB1*625、DPB1*626、DPB1*627、DPB1*628、DPB1*629、DPB1*63、DPB1*630、DPB1*631、DPB1*632、DPB1*633、DPB1*634、DPB1*635、DPB1*636、DPB1*637、DPB1*638、DPB1*639、DPB1*64、DPB1*640、DPB1*641、DPB1*642、DPB1*643、DPB1*644、DPB1*645、DPB1*646、DPB1*647、DPB1*648、DPB1*649、DPB1*65、DPB1*650、DPB1*651、DPB1*652、DPB1*653、DPB1*654、DPB1*655、DPB1*656、DPB1*657、DPB1*658、DPB1*659、DPB1*66、DPB1*660、DPB1*661、DPB1*662、DPB1*663、DPB1*664、DPB1*665、DPB1*666、DPB1*667、DPB1*668、DPB1*669、DPB1*67、DPB1*670、DPB1*671、DPB1*672、DPB1*673、DPB1*674、DPB1*675、DPB1*676、DPB1*677、DPB1*678、DPB1*679、DPB1*68、DPB1*680、DPB1*681、DPB1*682、DPB1*683、DPB1*684、DPB1*685、DPB1*686、DPB1*687、DPB1*688、DPB1*689、DPB1*69、DPB1*690、DPB1*691、DPB1*692、DPB1*693、DPB1*694、DPB1*695、DPB1*696、DPB1*697、DPB1*698、DPB1*699、DPB1*70、DPB1*700、DPB1*701、DPB1*702、DPB1*703、DPB1*704、DPB1*705、DPB1*706、DPB1*707、DPB1*708、DPB1*709、DPB1*71、DPB1*710、DPB1*711、DPB1*712、DPB1*713、DPB1*714、DPB1*715、DPB1*716、DPB1*717、DPB1*718、DPB1*719、DPB1*72、DPB1*720、DPB1*721、DPB1*722、DPB1*723、DPB1*724、DPB1*725、DPB1*726、DPB1*727、DPB1*728、DPB1*729、DPB1*73、DPB1*730、DPB1*731、DPB1*732、DPB1*733、DPB1*734、DPB1*735、DPB1*736、DPB1*737、DPB1*738、DPB1*739、DPB1*74、DPB1*740、DPB1*741、DPB1*742、DPB1*743、DPB1*744、DPB1*745、DPB1*746、DPB1*747、DPB1*748、DPB1*749、DPB1*75、DPB1*750、DPB1*751、DPB1*752、DPB1*753、DPB1*754、DPB1*755、DPB1*756、DPB1*757、DPB1*758、DPB1*759、DPB1*76、DPB1*760、DPB1*761、DPB1*762、DPB1*763、DPB1*764、DPB1*765、DPB1*766、DPB1*767、DPB1*768、DPB1*769、DPB1*77、DPB1*770、DPB1*771、DPB1*772、DPB1*773、DPB1*774、DPB1*775、DPB1*776、DPB1*777、DPB1*778、DPB1*779、DPB1*78、DPB1*780、DPB1*781、DPB1*782、DPB1*783、DPB1*784、DPB1*785、DPB1*786、DPB1*787、DPB1*788、DPB1*789、DPB1*79、DPB1*790、DPB1*791、DPB1*792、DPB1*794、DPB1*795、DPB1*796、DPB1*797、DPB1*798、DPB1*799、DPB1*80、DPB1*800、DPB1*801、DPB1*802、DPB1*803、DPB1*804、DPB1*805、DPB1*806、DPB1*807、DPB1*808、DPB1*809、DPB1*81、DPB1*810、DPB1*811、DPB1*812、DPB1*813、DPB1*814、DPB1*815、DPB1*816、DPB1*817、DPB1*818、DPB1*819、DPB1*82、DPB1*820、DPB1*821、DPB1*822、DPB1*823、DPB1*824、DPB1*825、DPB1*826、DPB1*827、DPB1*828、DPB1*829、DPB1*83、DPB1*830、DPB1*831、DPB1*832、DPB1*833、DPB1*834、DPB1*835、DPB1*836、DPB1*837、DPB1*838、DPB1*839、DPB1*84、DPB1*840、DPB1*841、DPB1*842、DPB1*843、DPB1*844、DPB1*845、DPB1*846、DPB1*847、DPB1*848、DPB1*849、DPB1*85、DPB1*850、DPB1*851、DPB1*852、DPB1*853、DPB1*854、DPB1*855、DPB1*856、DPB1*857、DPB1*858、DPB1*859、DPB1*86、DPB1*860、DPB1*861、DPB1*862、DPB1*863、DPB1*864、DPB1*865、DPB1*866、DPB1*867、DPB1*868、DPB1*869、DPB1*87、DPB1*870、DPB1*871、DPB1*872、DPB1*873、DPB1*874、DPB1*875、DPB1*876、DPB1*877、DPB1*878、DPB1*879、DPB1*88、DPB1*880、DPB1*881、DPB1*882、DPB1*883、DPB1*884、DPB1*885、DPB1*886、DPB1*887、DPB1*888、DPB1*889、DPB1*89、DPB1*890、DPB1*891、DPB1*892、DPB1*893、DPB1*894、DPB1*895、DPB1*896、DPB1*897、DPB1*898、DPB1*899、DPB1*90、DPB1*900、DPB1*901、DPB1*902、DPB1*903、DPB1*904、DPB1*905、DPB1*906、DPB1*907、DPB1*908、DPB1*909、DPB1*91、DPB1*910、DPB1*911、DPB1*912、DPB1*913、DPB1*914、DPB1*915、DPB1*916、DPB1*917、DPB1*918、DPB1*919、DPB1*92、DPB1*920、DPB1*921、DPB1*922, DPB1*923, DPB1*924, DPB1*925, DPB1*926, DPB1*927, DPB1*928, DPB1*929, DPB1*93, DPB1*930, DPB1*931, DPB1*932, DPB1*933, D PB1*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, DP DPB1*960, DPB1*961, DPB1*962, DPB1*963, DPB1*964, DPB1*965, DPB1*97, DPB1*98, and DPB1*99. In some embodiments, the DP beta chain comprises an HLA-DPB1*01, HLA-DPB1*02, HLA-DPB1*03, HLA-DPB1*04, HLA-DPB1*05, HLA-DPB1*06, HLA-DPB1*08, or HLA-DPB1*09 allele. In certain embodiments, the DP beta chain comprises an HLA-DPB1*04 allele. In certain embodiments, the DP beta chain comprises an HLA-DPB1*04:01 allele.

[0113] In certain embodiments, the MHC class II molecule comprises a DP beta chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO:3, wherein the DP beta chain comprises a tryptophan at the amino acid residue corresponding to position 112 of SEQ ID NO:1, and the DP beta chain comprises a methionine at the amino acid residue corresponding to position 141 of SEQ ID NO:1. In certain embodiments, an MHC class II molecule comprises a DP beta chain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO:3, wherein the DP beta chain comprises (i) a tryptophan at the amino acid residue corresponding to position 112 of SEQ ID NO:1, (ii) a methionine at the amino acid residue corresponding to position 141 of SEQ ID NO:1, (iii) a valine at the amino acid residue corresponding to position 114 of SEQ ID NO:1, and (iv) a methionine at the amino acid residue corresponding to the corresponding position 158 of SEQ ID NO:1. In certain embodiments, an MHC class II molecule comprises a DP beta chain comprising the amino acid sequence set forth in SEQ ID NO:3.

[0114] II.A.2. MHC class II alpha chain In some embodiments of the present disclosure, the MHC class II molecule further comprises an alpha chain. In some embodiments, the alpha chain is a wild-type alpha chain. In some embodiments, the alpha chain is a DP alpha chain. Any DP alpha chain may be used in the compositions and methods of the present disclosure. In some embodiments, the DP alpha chain comprises an HLA-DPA1*01, HLA-DPA1*02, HLA-DPA1*03, or HLA-DPA1*04 allele. In certain embodiments, the DP alpha chain comprises an HLA-DPA1*01 allele. In certain embodiments, the DP alpha chain comprises an HLA-DPA1*02 allele. In certain embodiments, the DP alpha chain comprises an HLA-DPA1*03 allele. In certain embodiments, the DP alpha chain comprises an HLA-DPA1*04 allele.

[0115] In certain embodiments, the DP alpha chain is DPA1*01:03:01:01, DPA1*01:03:01:02, DPA1*01:03:01:03, DPA1*01:03:01:04, DPA1*01:03:01:05, DPA1*01:03:01:06, DPA1*01:03:01:07, DPA1*01:03:01:08, DPA1*01:03:01:09, DPA1*01:03:01:10, DPA1*01:03:01:11, DPA1*01:03:01:12, DPA1*01:03:01:13, DPA1*01:03:01:14, DPA1*01:03:01:15, DPA1*01:03:01:16, DPA1*01:03:01:17, DPA1*01:03:01:18Q, DPA1*01:03:01:19, DPA1*01:03:01:20, DPA1*01:03:01:21, DPA1*01:03:01:22, DPA1*01:03:01:23, DPA1*01:03:02, DPA1*01:03:03, DPA1*01:03:04, DPA1*01:03:05, DPA1*01:03:06, DPA1*01:03:07, DPA1*01:03:08, DPA1*01:03:09, DPA1*01:04, DPA1*01:05, DPA1*01:06:01, DPA1*01:06:02, DPA1*01:07, DPA1*01:08, DPA1*01:09, DPA1*01:10, DPA1*01:11, DPA1*01:12, DPA1*01:13, DPA1*01:14, DPA1*01:15, DPA1*01:16, DPA1*01:17, DPA1*01:18, DPA1*01:19, DPA1*02:01:01:01, DPA1*02:01:01:02, DPA1*02:01:01:03, DPA1*02:01:01:04, DPA1*02:01:01:05, DPA1*02:01:01:06, DPA1*02:01:01:07, DPA1*02:01:01:08, DPA1*02:01:01:09, DPA1*02:01:01:10, DPA1*02:01:01:11, DPA1*02:01:02:01, DPA1*02:01:02:02, DPA1*02:01:03, DPA1*02:01:04, DPA1*02:01:05, DPA1*02:01:06,DPA1*02:01:07, DPA1*02:01:08:01, DPA1*02:01:08:02, DPA1*02:02:02:01, DPA1*02:02 :02:02, DPA1*02:02:02:03, DPA1*02:02:02:04, DPA1*02:02:02:05, DPA1*02:02:03, DPA 1*02:02:04, DPA1*02:02:05, DPA1*02:02:06, DPA1*02:03, DPA1*02:04, DPA1*02:05, DPA 1*02:06, DPA1*02:07:01:01, DPA1*02:07:01:02, DPA1*02:07:01:03, DPA1*02:08, DPA1* 02:09, DPA1*02:10, DPA1*02:11, DPA1*02:12, DPA1*02:13N, DPA1*02:14, DPA1*02:15, DP A1*02:16, DPA1*03:01:01:01, DPA1*03:01:01:02, DPA1*03:01:01:03, DPA1*03:01:01:0 4, 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.

[0116] II.A.3. Signal peptides In some embodiments, the DP beta chain and / or the DP alpha chain further comprise a signal peptide. Any signal peptide known in the art may be used in the compositions and methods disclosed herein. In some embodiments, the DP beta chain signal peptide is the same as the DP alpha signal peptide. In some embodiments, the DP beta chain signal peptide is different from the DP alpha signal peptide.

[0117] In some embodiments, the signal peptide is derived from a native signal peptide. In some embodiments, the signal peptide is derived from a naturally occurring DP beta chain signal peptide. In some embodiments, the signal peptide comprises a naturally occurring DP beta chain signal peptide. In some embodiments, the signal peptide is derived from a naturally occurring DP alpha chain signal peptide. In some embodiments, the signal peptide comprises a naturally occurring DP alpha chain signal peptide. In some embodiments, the signal peptide is derived from a fibroin light chain (FibL) signal peptide. In some embodiments, the signal peptide comprises SEQ ID NO:9. In some embodiments, the signal peptide is synthetic.

[0118] II.A.4. Transmembrane domains In some embodiments, the DP beta chain and / or DP alpha chain further comprise a transmembrane domain. The transmembrane domain can be of any length and of any origin. In some embodiments, the transmembrane domain is at least about 1 to at least about 50 amino acids in length. In some embodiments, the transmembrane domain is derived from a naturally occurring transmembrane domain. In some embodiments, the transmembrane domain comprises a naturally occurring transmembrane domain. In some embodiments, the transmembrane domain is derived from a naturally occurring HLA transmembrane domain. In some embodiments, the transmembrane domain comprises a naturally occurring HLA transmembrane domain. In some embodiments, the transmembrane domain is derived from a naturally occurring DP beta chain transmembrane domain. In some embodiments, the transmembrane domain comprises a naturally occurring DP beta chain transmembrane domain. In some embodiments, the transmembrane domain is derived from a naturally occurring DP alpha chain transmembrane domain. In some embodiments, the transmembrane domain comprises a naturally occurring DP alpha chain transmembrane domain.

[0119] II.A.5. Leucine zipper In some embodiments, the DP beta chain and / or DP alpha 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 embodiments, the DP beta chain and / or DP alpha chain comprises an acidic LZip (αLZip), a basic LZip (βLZip), or both. In some embodiments, one or more LZip sequences are derived from a naturally occurring LZip sequence. In some embodiments, one or more LZip sequences comprise a naturally occurring LZip sequence. In some embodiments, one or more LZip sequences are synthetic. In certain embodiments, one or more LZip sequences comprise the LZip sequence set forth in SEQ ID NO: 4 (Table 1).

[0120] II.A.6. Linkers In some embodiments, the DP beta chain and / or DP alpha chain useful in the present disclosure further comprises a linker. Any linker known in the art may be used in the compositions and methods disclosed herein. In certain embodiments, the linker comprises a Gly / Ser linker. In some embodiments, the linker comprises an amino acid sequence selected from GlySer, Gly2Ser, Gly3Ser, and Gly4Ser. In some embodiments, the linker is located at the N-terminus of the extracellular domain of the DP alpha chain or DP beta chain. In some embodiments, the linker is located at the C-terminus of the extracellular domain of the DP alpha chain or DP beta chain. In some embodiments, the linker is located between the extracellular domain and the transmembrane domain of the DP alpha chain or DP beta chain. In some embodiments, the linker is located between the extracellular domain of the DP alpha chain or DP beta chain and one or more LZip sequences. In some embodiments, the linker is located between the extracellular domain of the DP alpha chain or DP beta chain and the signal peptide.

[0121] Linkers of any length may be used in the compositions and methods disclosed herein. In some embodiments, the linker is at least one amino acid in length. In some embodiments, 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, or at least about 1 to at least about 3 amino acids in length.

[0122] In some embodiments, 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 length. In certain embodiments, the linker is about 3 amino acids in length. In certain embodiments, the linker is about 4 amino acids in length. In certain embodiments, the linker is about 5 amino acids in length.

[0123] II.B. Cells In certain embodiments of the present disclosure, the MHC class II molecule of the present disclosure is linked or bound to the membrane of a cell. In certain embodiments, the beta chain of the MHC class II molecule is linked or bound to the membrane of a cell. In certain embodiments, the alpha chain of the MHC class II molecule is linked or bound to the membrane of a cell. In certain embodiments, the alpha and beta chains of the MHC class II molecule are linked or bound to the membrane of a cell.

[0124] Certain embodiments of the present disclosure relate to cells comprising the MHC class II molecules disclosed herein. Any cells may be used in the compositions described herein. In certain embodiments, the cells are mammalian cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are derived from healthy cells, such as healthy fibroblasts. In some embodiments, the cells are derived from tumor cells. Non-limiting examples of cells useful in the present 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 certain embodiments, the cells are any cells disclosed in Hasan et al., Adv. Genet. Eng. 4(3):130 (2015), incorporated herein by reference.

[0125] In certain embodiments, the cell is a professional APC. In certain embodiments, the cell is a macrophage, a B cell, a dendritic cell, or any combination thereof.

[0126] In certain embodiments, the cells lack endogenous expression of one or more MHC class II alleles. In some embodiments, the cells lack endogenous expression of HLA-DP alleles. In some embodiments, the cells lack endogenous expression of HLA-DP alpha chain alleles. In some embodiments, the cells lack endogenous expression of HLA-DP beta chain alleles.

[0127] II.C. Soluble MHC class II molecules In certain embodiments, the MHC class II molecule is not bound to a cell membrane, for example, the MHC class II molecule is in a soluble form. As used herein, a soluble MHC class II molecule includes any MHC class II molecule or a portion thereof described herein, and is not bound to a cell membrane. In certain embodiments, the MHC class II molecule or a portion thereof is not bound to any membrane. In some embodiments, the MHC class II molecule or a portion thereof is bound to an inert particle. In some embodiments, the MHC class II molecule or a portion thereof is bound to the membrane of an extracellular vesicle. In some embodiments, the MHC class II molecule is bound to an artificial membrane or an artificial surface, for example, the surface of an array plate.

[0128] Any inert particle known in the art may be used in the compositions and methods of the present disclosure. In some embodiments, the inert particle is a bead. In some embodiments, the bead is a glass bead, a latex bead, a metal bead, or any combination thereof. In some embodiments, the inert particle is a nanoparticle (NP). Any NP known in the art may be used in the compositions and methods of the present disclosure. In certain embodiments, the nanoparticle is selected from PEGylated iron oxide, chitosan, dextran, gelatin, alginate, liposomes, starch, branched polymers, carbon-based carriers, polylactic acid, poly(cyano)acrylate, polyethyleinemine, block copolymers, polycaprolactone, SPIONS, USPIONS, Cd / Zn-selenide, or silica nanoparticles. In certain embodiments, the nanoparticle is a PEGylated iron oxide nanoparticle. Non-limiting examples of nanoparticles useful in the compositions and methods disclosed herein include those described in De Jong and Borm, Int. J. Nanomedicine 3(2):133-49 (2008), and Umeshappa et al., Nat. Commun. 10(1):2150 (May 14, 2019), each of which is incorporated herein by reference in its entirety.

[0129] In some embodiments, the MHC class II molecule comprises a fragment of a full-length MHC class II molecule, wherein one or more amino acids of the transmembrane domain of the alpha chain and / or the transmembrane domain of the beta chain are deleted. In some embodiments, the MHC class II molecule comprises the extracellular domain of the alpha chain (e.g., as set forth in SEQ ID NO:6) and / or the extracellular domain of the beta chain (e.g., as set forth in SEQ ID NO:1 or 3). In certain embodiments, the MHC class II molecule comprises a DP alpha chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:6. In some embodiments, the MHC class II molecule comprises a DP alpha chain comprising the amino acid sequence set forth in SEQ ID NO:6.

[0130] In certain embodiments, the MHC class II molecule comprises a DP beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 1. In some embodiments, the MHC class II molecule comprises a DP beta chain comprising an amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the MHC class II molecule comprises a DP beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 3. In some embodiments, the MHC class II molecule comprises a DP beta chain comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the MHC class II molecule comprises a DP beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 4. In some embodiments, the MHC class II molecule comprises a DP beta chain comprising an amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the MHC class II molecule comprises a DP beta chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 5. In some embodiments, the MHC class II molecule comprises a DP beta chain comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0131] II.D. Nucleic Acid Molecules and Vectors Certain aspects of the present disclosure relate to nucleic acid molecules encoding the MHC class II molecules disclosed herein. In some aspects, the nucleic acid molecule encodes the MHC class II beta chain disclosed herein. In certain aspects, the nucleic acid molecule encoding the MHC class II beta chain comprises a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:2.

[0132] In some embodiments, the nucleic acid molecule encodes an MHC class II alpha chain disclosed herein. In certain embodiments, the nucleic acid molecule encoding the MHC class II alpha chain comprises a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:7.

[0133] In some embodiments, the nucleic acid molecule encodes both an MHC class II alpha chain as disclosed herein and an MHC class II beta chain as disclosed herein. In some embodiments, the sequence encoding the MHC class II alpha chain is under the control of the same promoter as the sequence encoding the MHC class II beta chain. In some embodiments, the sequence encoding the MHC class II alpha chain is under the control of a first promoter and the sequence encoding the MHC class II beta chain is under the control of a second promoter.

[0134] In some aspects, the present disclosure relates to a first nucleic acid molecule encoding an MHC class II beta chain as disclosed herein, and a second nucleic acid molecule encoding an MHC class II alpha chain as disclosed herein.

[0135] Certain aspects of the present disclosure relate to a vector or set of vectors comprising a nucleic acid molecule disclosed herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a viral particle or virus. In some embodiments, the vector is a mammalian vector. In some embodiments, the vector is a bacterial vector.

[0136] In certain embodiments, the vector is a retroviral vector. In some embodiments, the vector is an adenoviral vector, a lentivirus, a Sendai virus, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a cowpox virus vector, a herpes simplex virus vector, or an adeno-associated virus (AAV) vector. In certain embodiments, the vector is an AAV vector. In some embodiments, the vector is a lentivirus. In certain embodiments, the vector is an adenoviral vector. In some embodiments, the vector is a Sendai virus. In some embodiments, the vector is a hybrid vector. For examples of hybrid vectors that can be used in the present disclosure, see Huang and Kamihira, Biotechnol. Adv. 31(2):208-23 (2103), which is incorporated herein by reference.

[0137] III. Methods of the Disclosure Certain aspects of the present disclosure relate to methods of treating a disease or condition in a subject. In some aspects, the present disclosure relates to methods of enhancing an immune response in a subject in need thereof.

[0138] III.A. Tumor Treatment Methods Certain aspects of the present disclosure relate to a method of treating cancer in a subject in need thereof, the method 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.

[0139] In some embodiments, the cancer is melanoma, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or intraocular malignant melanoma, pancreatic cancer, skin cancer, head and neck cancer, 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 intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue cancer, The cancer is selected from sarcoma, urethral cancer, penile cancer, chronic or acute 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) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those due to asbestos, other B-cell malignancies, and combinations of cancers. In some embodiments, the cancer is melanoma.

[0140] In some embodiments, the cancer is recurrent. In some embodiments, the cancer is refractory. In some embodiments, the cancer is progressive. In some embodiments, the cancer is metastatic.

[0141] In some embodiments, the methods disclosed herein treat cancer in a subject. In some embodiments, the methods disclosed herein reduce the severity of one or more symptoms of cancer. In some embodiments, the methods disclosed herein reduce the size or number of tumors resulting from cancer. In some embodiments, the methods disclosed herein extend the overall survival of a subject compared to a subject not provided with a method disclosed herein. In some embodiments, the methods disclosed herein extend the progression-free survival of a subject compared to a subject not provided with a method disclosed herein. In some embodiments, the methods disclosed herein result in a partial response in a subject. In some embodiments, the methods disclosed herein result in a complete response in a subject.

[0142] Certain aspects of the present disclosure relate to methods of treating an infection in a subject in need thereof, the method 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 embodiments, the virus is herpesvirus, HIV, papovavirus, measles virus, rubella virus, human papillomavirus (HPV), human T-lymphotropic virus 1, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, norovirus, and any combination thereof. In some embodiments, the bacterium is selected from Streptococcus, Staphylococcus, and E. coli. In some embodiments, the bacterial infection is selected from brucellosis, Campylobacter infection, cat scratch disease, cholera, Escherichia coli, gonorrhea, Klebsiella, Enterobacter, Serratia, Legionella infection, meningococcal infection, whooping cough, plague, Pseudomonas infection, Salmonella infection, shigellosis, typhoid fever, tularemia, splenic ulcers, diphtheria, enterococcal infection, erysipelothricosis, listeriosis, nocardiosis, pneumococcal infection, staphylococcal infection, streptococcal infection, and any combination thereof. In some embodiments, the parasitic infection is selected from pinworm, trichomoniasis, toxoplasmosis, giardiasis, cryptosporidiosis, malaria, hookworm, ringworm, tapeworm, fluke, and any combination thereof. In some embodiments, the fungal infection is selected from Candida, Malassezia furfur, dermatophytes (eg, Epidermophyton, Microsporum, and Trichophyton), or any combination thereof.

[0143] In some embodiments, the methods disclosed herein include treating cancer or an infection in a subject in need thereof, the methods comprising administering to the subject a cell described herein, wherein the cell comprises an MHC class II molecule disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, or any combination thereof.

[0144] In some embodiments, the cells are obtained from the subject. In some embodiments, the cells are obtained from a donor other than the subject.

[0145] III.B. Methods for Enriching Target Populations of T Cells Certain embodiments of the present disclosure relate to methods for enriching a target population of T cells obtained from a human subject. In some embodiments, the method comprises contacting the T cells with an HLA class II molecule disclosed herein. In some embodiments, the method comprises contacting the T cells with a cell, e.g., an APC, disclosed herein. In some embodiments, after contacting, the enriched population of T cells comprises a greater number of T cells capable of binding to HLA class II molecules compared to the number of T cells capable of binding to HLA class II molecules before contacting.

[0146] Some embodiments of the present disclosure relate to methods for selecting T cells capable of targeting diseased cells, e.g., tumor cells. In some embodiments, the methods include contacting a population of in vitro isolated T cells with a complex comprising an MHC class II molecule disclosed herein and a fragment of a polypeptide, e.g., an antigen expressed on diseased cells, e.g., a tumor-expressed polypeptide, e.g., an epitope. In some embodiments, the T cells are obtained from a human subject.

[0147] The T cells obtained from the human subject can be any T cells disclosed herein. In some embodiments, the T cells obtained from the human subject are tumor-infiltrating lymphocytes (TILs).

[0148] In some embodiments, the method further comprises administering the enriched T cells to a human subject. In some embodiments, the subject is pre-conditioned prior to receiving the T cells as described herein.

[0149] Any of the various aspects, embodiments and options described herein can be combined in any and all variations.

[0150] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0151] Having generally described the present disclosure, a further understanding can be obtained by reference to the examples provided herein, which are for illustrative purposes only and are not intended to be limiting. [Example]

[0152] Example 1 - Method

[0153] cell Peripheral mononuclear cells were obtained by density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare Life Sciences, Marlborough, MA). The K562 cell line is an erythroleukemia cell line deficient in HLA class I / II expression. K562-based artificial APCs (aAPCs) expressing various HLA class II genes individually as single HLA alleles related to CD80 and CD83 have been previously reported (Butler et al., PloS One 7, e30229 (2012)). The Jurkat76 cell line is a T-cell leukemia cell line lacking endogenous TCR, CD4, and CD8 expression. Jurkat76 / CD4 cells were generated by retroviral transduction with 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 (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS and 50 μg / ml gentamicin. K562 and Jurkat76 cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 50 μg / ml gentamicin.

[0154] peptide Synthetic peptides were purchased from Genscript (Piscataway, NJ) and dissolved in DMSO at 50 μg / ml. Peptide sequences are shown in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0155] gene The novel TCR genes were cloned by rapid amplification of 5' cDNA ends (RACE) PCR using the SMARTer RACE 5' / 3' Kit (Takara Bio, Shiga, Japan) and sequenced as described above. All genes were cloned into pMX retroviral vectors and transduced into cell lines using the 293GPG and PG13 cell-based retroviral systems.

[0156] antibody For flow cytometry analysis, PE-conjugated anti-class II (9-49(I3)), APC-Cy7-conjugated anti-CD4 (RPA-T4, Biolegend, San Diego, CA) 44 The following antibodies were used: 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). 157-170 and DP4 / WT1 329-348 Monomers were multimerized using PE-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Dead cells were sorted using the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit 465 (Thermo Fisher Scientific, Waltham, MA). Stained cells were analyzed using a Canto II or LSRFortessa X-20 (BD Biosciences, Franklin Lakes, NJ). Cell sorting was performed using a FACS Aria II (BD Biosciences, Franklin Lakes, NJ). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR).

[0157] For immunoblot analysis, the following antibodies were used: 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).

[0158] TCR transduction of primary T cells CD3 + T cells and CD4 + T cells were isolated using a Pan T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD4 + The T cells were purified using a T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified T cells were stimulated with aAPC / mOKT3 irradiated at 200 Gy at an E:T ratio of 20:1. Starting the following day, activated T cells were retrovirally transduced with the cloned TCR gene by centrifugation at 1,000 × g for 1 hour at 32°C or by using Retronectin-coated plates (Takara Bio, Shiga, Japan) for 3 consecutive days. The following day, 100 IU / ml IL-2 and 10 ng / ml IL-15 were added to the TCR-transduced T cells. The culture medium was replenished every 2–3 days.

[0159] Staining with soluble CD4 The soluble CD4 (sCD4) gene was generated by fusing the human CD4 extracellular domain with a 6xHis tag via a GS linker. The sCD4 gene was retrovirally transduced into HEK293T cells, and the culture supernatant containing sCD4 monomers was collected. The sCD4 was dimerized with a PE-labeled anti-6xHis tag mAb (AD1.1.10, Abcam, Cambridge, MA) and used. HLA class II-expressing K562 cells were stained with dimerized sCD4 in the presence of goat serum at room temperature for 30 minutes. Surface HLA class II expression on K562-derived cells expressing various class II genes individually is shown in Figures 13A-13Q.

[0160] Construction and screening of an intralocus multipoint-induced DPB1*04:01 mutant cDNA library Intralocus multipoint random mutations were inserted into DPB1*04:01 cDNA by PCR using the following primer sets: forward: 5'-CACCACAACNNNCTTNNNTGCCACGTG-3' (SEQ ID NO: 12) and reverse: 5'-CACGTGGCANNNAAGNNNGTTGTGGTG-3' (SEQ ID NO: 13) for L112 and V114; forward: 5'-ACAGCTGGGGTCNNNTCCACCAACCTG-3' (SEQ ID NO: 14) and reverse: 5'-CAGGTTGGTGGANNNGACCCCAGCTGT-3' (SEQ ID NO: 15) for V141; forward: 5'-CAGATCNNNGTGNNNCTGGAAATGACC-3' (SEQ ID NO: 16) and reverse: 5'-GGTCATTTCCAGNNNCACNNNGATCTG-3' (SEQ ID NO: 17) for L156 and M158. N denotes any nucleotide. The resulting PCR fragments were fused to each other to construct a mutant full-length DPB1*04:01 cDNA expression library with random mutations at positions L112, V114, V141, L156, and M158. K562 cells stably expressing the DPA1*01:03 gene were infected with recombinant retroviruses generated using 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 a flow cytometry cell sorter. The mutant DPB1*04:01 gene was cloned from the collected cells and retrovirally transduced into K562 cells along with the wild-type DPA1*01:03 gene as described above.

[0161] Formation of HLA class II monomers and dimers The extracellular domain of the wild-type class II α gene was fused to an acidic leucine zipper via a GGGS linker, followed by a 6xHis tag via a GS linker (see SEQ ID NO: 8). The extracellular domain of the mutated class II β gene (see SEQ ID NO: 3) was 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 revealed soluble DP4. L112W / V141M HEK293T cells stably secreting proteins were grown to confluence, and the medium was replaced with serum-free 293 SFM II medium (Thermo Fisher Scientific, Waltham, MA). After 48 h, the conditioned medium was collected and concentrated using an Amicon Ultra filter (10 kDa molecular weight cut-off (MWCO)) (MilliporeSigma, Burlington, MA). The supernatant containing soluble HLA class II was then mixed with 100 μg / ml of the peptide of interest for 20–24 h at 37°C for in vitro peptide exchange. Monomers without peptide exchange were used as controls. The concentration of the monomer was measured by specific ELISA using nickel-coated plates (XPressBio, Frederick, MD) and an anti-His tag biotinylated mAb (AD1.1.10, R&D Systems, Minneapolis, MN). Soluble HLA class II monomers were dimerized with PE-conjugated anti-Hism Ab (AD1.1.10, Abcam, Cambridge, MA) at a molar ratio of 2:1 for 1.5 hours at 4°C for staining.

[0162] DP4-restricted antigen-specific CD4 + T cell stimulation CD4 + T cells, CD4 +CD4 T cells were purified using a T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified T cells were stimulated with DP4-expressing aAPCs pulsed with 10 μg / ml of DP4-restricted peptide and irradiated at 200 Gy at an E:T ratio of 20:1. After 48 hours, 10 IU / ml of IL-2 and 10 ng / ml of IL-15 were administered to CD4 T cells. + The T cells were then added to the culture medium supplemented with IL-2 (10 IU / ml) and IL-15 (10 ng / ml) every 2–3 days. After 2 weeks of stimulation, the T cells were incubated with DP4 L112W / V141M The cells were subjected to dimer staining.

[0163] HLA class II dimer and tetramer staining Primary T cells transduced with exogenous TCR genes and Jurkat76 / CD4 T cells were pretreated with 50 nM dasatinib (LC Laboratories, Woburn, MA) for 30 min at 37°C and stained with 5–15 μg / ml class II dimers at room temperature for 4–5 h. 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.

[0164] ELISPOT assay Cytokine ELISPOT assays were performed as previously reported (see, e.g., Yamashita et al., Nat. Commun. 8:15244 (2017) and Anczurowski et al., Sci. Rep. 8:4804 (2018)).

[0165] Immunoblotting Immunoblot analysis was performed as previously reported (see, e.g., Yamashita et al., Nat. Commun. 8:15244 (2017) and Anczurowski et al., Sci. Rep. 8:4804 (2018)).

[0166] Protein modeling The HLA-DP4 and human CD4 complex model structures were predicted based on the structures of PDB IDs: 3S5L and 3T0E using the Swiss-Model workspace for four-component structure prediction.

[0167] statistical analysis Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA). An unpaired two-tailed Student's t-test was used for two-sample comparison. No statistical methods were used to predetermine sample size. Researchers were aware of allocation during the experiment or evaluation of results. The experiment was not randomized.

[0168] Biolayer interferometry sensorgram The extracellular domain of human CD4 (residues 26-440 of NP_000607.1), followed by a GS linker and a 10x His tag, was stably expressed in the human cell line A375 (SEQ ID NOs: 245-246; Table 4). 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 a 10 kDa MWCO Amicon Ultra-15 spin column (MilliporeSigma, Burlington, MA). The buffer was exchanged into HBS-EP (GE Healthcare Life Sciences, Marlborough, MA) using a 10 kDa MWCO MINI Dialyzer (Thermo Fisher Scientific, Waltham, MA). The purity of the recombinant CD4 protein was consistently greater than 90%, as confirmed by SDS-PAGE.

[0169] The recombinant DP4 protein consisted of the extracellular domain of DPA1*01:03 and either wild-type DPB1*04:01 or the L112W / V141M mutant. DPA1*01:03 was followed by an acidic leucine zipper, a GS linker, and a 10x histidine tag, while wild-type and mutant DPB1 were followed by a basic leucine zipper, a GS linker, and a biotinylation sequence (GLNDIFEAQKIEWHE; SEQ ID NO: 244). Both the DPA and DPB genes were stably expressed in A375-BirA cells, which were transfected with a codon-optimized BirA gene encoding a leader sequence at the 5' end and an ER-retention KDEL motif at the 3' end. The recombinant DP4 protein was purified from the supernatant using TALON metal affinity resin (Takara Bio, Shiga, Japan). The eluted protein was concentrated using a 10 kDa MWCO Vivaspin 500 spin column (GE Healthcare Life Sciences, Marlborough, MA) and reconstituted to a working volume in PBS.

[0170] Wild-type DP4 and DP4 L112W / V141M Binding of DP4 to CD4 was measured using the Octet Red system (ForteBio, Fremont, CA). Experiments were performed at 25°C with constant shaking at 1,000 rpm in a 96-well plate (Perkin Elmer, Waltham, MA) with a sample volume of 200 μl. Biotinylated recombinant DP4 was loaded onto a streptavidin-coated biosensor (ForteBio, Fremont, CA) to saturation, followed by a baseline measurement in HBS-EP buffer. Binding was measured by incubating the sensor with titrated concentrations of recombinant CD4 (0.8125–26 μM) for 400 s, followed by dissociation in HBS-EP buffer alone for 300 s. Steady-state analysis was fitted using a one-site specific binding model in GraphPad Prism 7.0. [Table 4]

[0171] Example 2 - Enhanced binding of DP to CD4 by L112W / V141M substitutions in the DP β chain A cDNA expression library of the DPB1*04:01 (DP4β) gene with random mutations at L112, V114, V141, L156, and M158 (corresponding to L114, V116, V143, L158, and M160 of the DR1 β chain, respectively) was generated, and the library was coexpressed with the wild-type DPA1*01:03 (DPα) gene in class II-deficient K562 cells. After two rounds of screening using soluble CD4 protein (sCD4), a cell population with enhanced CD4 binding was isolated, from which mutant DP4β genes with L112W, V114M, V141M, and M158I substitutions were molecularly cloned. When ectopic expression was performed in K562 cells, the wild-type DPα chain and the mutant DP4β genes with L112W, V114M, V141M, and M158I substitutions (DP4β) were expressed. L112W / V114M / V141M / M158I Mutant DP4 molecules composed of a cloned mutant DP4 β chain with β-reducing ...

[0172] To determine which of the four mutations was important for the enhanced CD4 binding, we performed a reversion mutagenesis study. All possible reversion DP4 mutants were reconstituted on class II-negative K562 cells and stained with sCD4. Both L112W and V141M (but not the V114M single substitution or the M158I single substitution) individually enhanced DP4 binding to sCD4 (Figure 1G). Importantly, the L112W / V141M double mutation (DP4 L112W / V141M ) synergistically enhanced DP4 / CD4 binding (Fig. 1G). Interestingly, both the V114M single substitution and the M158I single substitution enhanced DP4 / CD4 binding. L112W / V141M This mutation appeared to negatively impact the enhanced binding potential (Figure 1G). Previous studies have shown that the K binding between CD4 and HLA class II DThe value has been estimated to be greater than 2 mM. Using a biolayer interferometry (BLI) binding assay, DPR L112 / V141M The affinity of DP4 to CD4 was measured. No binding was detected between wild-type DP4 and CD4, but DP4 L112W / V141M is K D DP4 bound to CD4 with a binding affinity of 8.9 μM (±1.1) (Fig. 1H and Fig. 1X-1BK). This value represents at least a 200-fold improvement in binding affinity. Furthermore, the binding affinity between CD4 and DP4 L112W / V141M The affinity observed between DP4 is higher than that between human CD8 and HLA class I (approximately 200 μM) and is comparable to that between mouse CD8 and mouse MHC class I (approximately 10 μM). L112W / V141M Enhanced binding between CD4 and CD4 + To confirm that this resulted in an enhanced T cell response, DP4 / WT1 TCR (clone 9)-transduced CD4 - and CD4 + Jurkat76T cells were used to express wild-type DP4 or DP4 as a single class II allele. L112W / V141M We compared the immune activation abilities of artificial APCs (aAPCs) expressing DP4. L112W / V141M It was demonstrated that aAPCs bearing α-APCs exhibited enhanced T cell stimulatory activity in a CD4-dependent manner (Figure 1I).

[0173] Next, we analyzed other DP alleles similarly to determine the enhanced binding of the L112W / V141M mutation to CD4. While neither wild-type DP2, DP5, nor DP8 bound to CD4, when the L112W / V141M double mutation was introduced into the DP β chain of these molecules, all three molecules strongly bound to CD4 (Figures 1J-1W). L112W / V141M Based on previous reports that revealed that the L112W / V141M mutation induces hydrophobic effects at positions K35, Q40, and T45 of CD4 in the CD4 complex, we constructed a structural model (Figures 2A-2D). These results indicated that the L112W / V141M mutation can enhance CD4 binding of at least four of the DP alleles tested.

[0174] Example 3 - Affinity matured DP4 L112W / V141M Specific staining of cognate TCRs with multimers To determine the effect of the L112W / V141M double mutation in DP4β on DP4 multimer staining, we used soluble DP4 L112W / V141M Monomers were generated, which were then dimerized with anti-His tag mAb. Three different DP4-restricted TCRs specific for MAGE-A3 (clone R12C9), WT1 (clone 9), and NY-ESO-1 (clone 5B8) were individually transduced into primary T cells, followed by cognate DP4. L112W / V141M As shown in Figures 3A-3P, each DP4 L112W / V141M Dimeric CD4 expressing the cognate TCR + T cells were specifically stained. R12C9 and clone 9 transduced T cells were stained with the corresponding DP4 L112W / V141M Co-staining with the dimers and anti-Vβ22 mAb and anti-NGFR mAb, respectively, revealed that all TCR-transduced CD4 + T cells express the corresponding DP4 L112W / V141M Successful staining of the dimer was visually confirmed (Figures 4A to 4H). Compared with the conventional wild-type DP4 tetramer, the novel DP4 of the present invention L112W / V141M The dimer stained both DP4 / WT1 and DP4 / NY-ESO-1 T cells better than the conventional wild-type DP4 tetramer (Figures 5A-5P). Notably, the conventional wild-type DP4 / NY-ESO-1 tetramer failed to stain cognate T cells even at effective high concentrations (data not shown).

[0175] Example 4 - Robust and versatile DP4 L112W / V141M dimer technology DP4 L112W / V141M To determine the robustness and versatility of multimer staining, we performed an extensive screening of potential DP4-restricted peptides derived from a series of tumor-associated antigens for their in vitro immunogenicity (Table 3). 196 DP4-restricted and tumor-associated antigen-derived 20-mer peptides were predicted using a peptide prediction algorithm (NetMHC2 ver. 2.2) and chemically synthesized (Table 3). Antigen-specific CD4+ T cell frequencies are generally very low in the periphery. + Primary CD4 isolated from melanoma patients + T cells were stimulated only once with each of the 196 peptide-pulsed DP4-aAPCs, and cognate DP4 L112W / V141M Dimer staining was performed. To avoid potential in vitro priming, mild stimulation conditions were utilized. As shown in Figures 6A-6F, 103 predicted DP4 peptides were immunogenic, at least in vitro.

[0176] To verify the dimer staining results, we isolated CCND1 from dimer-positive T cells. 219-238 , HSD17B12 225-244 , LGSN 296-315 , MAGE-A2 108-127 , and MUC5AC 4922-4941 Seven DP4-restricted TCR genes specific for human CD4 were cloned (Figures 7A-7L and Table 5). + When clonotypically rearranged in TCR-deficient T cells, all of these TCRs expressed the cognate DP4 L112W / V141M The dimer stained normally (Figures 8A-8X) and functioned in a DP4-restricted and antigen-specific manner (Figures 9A-9G).

[0177] Of the four TCRs individually expressed on primary T cells, three TCRs, namely 03-CCND1 219-238 , 06-MAGE-A2 108-127 and 05-MUC5AC 4922-4941 was able to recognize the cognate peptide endogenously processed and presented by DP4 (Figures 10A-10Q and 11A-11E). Importantly, 06-MAGE-A2 108-127 Transduced primary T cells were able to recognize melanoma cell lines in a DP4- and MAGE-A2-dependent manner (FIGS. 12A-12E). [Table 5]

[0178] In contrast to CD8, the role and function of CD4 as a coreceptor have not yet been fully elucidated. This lack of information is mainly due to the very weak binding of CD4 to class II, which severely limits research into the role of the relationship between CD4 and class II. In this study, we investigated the affinity-matured form of HLA-DP4 with enhanced binding to CD4, i.e., DP4 L112W / V141M and isolated a novel DP4 L112W / V141M We developed a dimer technology that allows the generation of DP4-restricted antigen-specific CD4 + It provides robustness and precision in detecting T cells.

[0179] This DP4 L112W / V141M Using dimer technology, we comprehensively investigated DP4-restricted antitumor T cell responses in vitro and identified multiple DP4-restricted immunogenic peptides and cognate TCR genes. HLA-DP4 is the most prevalent HLA allele in many ethnic groups, and DP4 is the most prevalent HLA allele in many ethnic groups. 84Gly Unlike other class II molecules, DP4 and other DP 84Gly The molecule constitutively presents peptides derived from endogenous sources, regardless of invariant chain and HLA-DM expression. Improved presentation of endogenous peptides by class II correlates with prolonged survival in cancer patients. In particular, the first human class II-restricted TCR gene therapy actually targeted the DP4-restricted MAGE-A3 peptide (see, e.g., Yao et al., J. Immunother. 39:191-201 (2016)). DPs such as DP2 and DP4 84Gly Genotypes act as risk alleles for antineutrophil cytoplasmic autoantibody-associated vasculitis. DP4 molecules, which can constitutively present peptides derived from endogenous tumor-associated antigens, may induce clinically relevant antitumor responses more than other class II molecules and may function as protective class II alleles.

[0180] For the identification of affinity-matured class II molecules, this example details several mutations in the β chain, but not the α chain, since the β chain interacts more directly with CD4 than the α chain. It is possible that additional mutations in the α and / or β chains may further enhance the binding of class II to CD4. However, the use of soluble class II molecules with excess CD4-binding capacity may be necessary to further enhance the binding of CD4. + This can lead to non-specific staining of T cells, which can have deleterious effects.

[0181] In conclusion, CD4 + T cells play a key role in the development of autoimmune diseases and in the defense against pathogenic infections and cancer. The novel HLA class II multimer technology described herein allows the synthesis of HLA class II-restricted CD4 T cells across HLA-DP alleles. + This can better facilitate the study of T cell responses.

[0182] Example 5-DP4 L112W / V141M Specificity and Binding Endogenous (non-transduced) antigen-specific CD4 + DP4 multimer staining of T cells was analyzed. L112W / V141M The dimer (Figures 14A-14B) inhibited endogenous TRPC1 more potently than the conventional DP4 dextramers (Figures 14C-14D). 578-597 specific CD4 + T cells were positively stained. L112W / V141M The dimer significantly reduced the expression of endogenous (non-transduced) NY-ESO-1 compared to the conventional tetramer (Figures 15C-15D) or dextramer (Figures 15E-15F). 157-170 specific CD4 + It was shown to significantly improve staining of T cells (Figures 15A-15B; Table 6). [Table 6-1] [Table 6-2]

[0183] Next, without in vitro stimulation, we expressed DP4 specific for a series of pathogen-associated peptides. L112W / V141M Dimeric memory CD4 + Ex vivo staining of T cells was performed. Tetanus toxin 948-968 (TT 948-968 ), herpes simplex virus type 2-UL21 283-302 (HSV-2-UL21 283-302 ), and respiratory syncytial virus glycoprotein 162-175 (RSV-GP 162-175 ) for DP4 L112W / V141M CD4 dimers + A small subset of T cells stained positive (Figures 16A-16Y). Next, we investigated the RSV-GP 162-175 (Figures 17A to 17V), and TT 948-968 Dimer (Figures 18A-18R) + CD4 + Endogenous (non-transduced) single-cell clones were constructed from T cells by limiting dilution. These T cell clones produced IL-2 in an antigen-specific manner (Figs. 17W and 18S). Multiple TCRαβ pairs, including one dominant pair, were transduced with DP4. L112W / V141M RSV-GP and TT dimers + In Figures 17A-17W and 18A-18S, the single cell clones were isolated from RSV-GP 162-175 and T.T. 948-968 dimer + These RSV-GP and TT dimers were constructed by limiting dilution from cells. + Single cell clones were cultured with three different DP4 multimers (DP4 L112W / V141M DP4 was stained individually with DP4 dimer, wild-type DP4 tetramer, or wild-type DP4 dextramer. L112W / V141M The dimers were shown to stain RSV-GP-specific clones (c12 and c39) and TT-specific clones (c2 and c9) better than the conventional wild-type DP4 RSV-GP dextramers and the wild-type DP4 TT tetramer and dextramers (Figures 19A-19NN).

[0184] method cell Peripheral mononuclear cells were obtained by density gradient centrifugation. K562-based artificial antigen-presenting cells (aAPCs) expressing various HLA class II genes individually as single HLA alleles associated with CD80 and CD83 have been previously reported (Butler, MO et al., PLoS One 7, e30229 (2012)). HEK293T cells were grown in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin.

[0185] Peptides / Antibodies The synthetic peptides were dissolved in DMSO at 50 μg / ml. APC-Cy7-conjugated anti-CD4 and PE-conjugated anti-His tag antibodies were used for flow cytometry analysis.

[0186] Formation of HLA class II monomers and dimers HEK293T cells were transfected with the α and β genes using a 293GPG cell-based retroviral system (see Hirano, N. et al., Blood 107, 1528-1536 (2006); Butler, MO et al., Clin Cancer Res 13, 1857-1867 (2007); Hirano, N. et al., Blood 108, 2662-2668 (2006)) and cultured in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin. DP4 dimer staining revealed soluble DP4. L112W / V141MHEK293T cells stably secreting proteins were grown to confluence, and the medium was replaced with serum-free 293 SFM II medium (Thermo Fisher Scientific, Waltham, MA). After 48 h, the conditioned medium was collected and concentrated using an Amicon Ultra filter (10 kDa molecular weight cut-off (MWCO)) (MilliporeSigma, Burlington, MA). The supernatant containing soluble HLA class II was then mixed with 100 μg / ml of the peptide of interest for 20–24 h at 37°C for in vitro peptide exchange. The concentration of the monomer was measured by specific ELISA using nickel-coated plates and an anti-His tag biotinylated mAb. For staining, the soluble HLA class II monomer was dimerized with a PE-conjugated anti-Hism Ab at a 2:1 molar ratio for 1.5 h at 4°C.

[0187] DP4-restricted antigen-specific CD4 + T cell stimulation CD4 + T cells were purified and stimulated with DP4-expressing aAPCs pulsed with 10 μg / ml of DP4-restricted peptide and irradiated at 200 Gy with an E:T ratio of 20:1. After 48 hours, 10 IU / ml of IL-2 and 10 ng / ml of IL-15 were added to the CD4+ T cells. 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, T cells were stimulated with DP4. L112W / V141M The cells were subjected to dimer staining.

[0188] HLA class II dimer, tetramer, and dextramer staining DP4 tetramer and dextramer were compared in a multimer staining analysis. Primary CD4 transduced with antigen-specific TCR genes + T cells were pretreated with 50 nM dasatinib for 30 min at 37°C and stained with 5–15 μg / ml class II dimers for 4–5 h at room temperature. After washing, cell surface molecules were counterstained with APC-Cy7-conjugated anti-CD4 mAb.

[0189] Unstimulated CD4 from PBMCs from melanoma patients + Dimer staining of T cells 1 million CD4 + Purified T cells were pretreated with 50 nM dasatinib for 30 min at 37°C. Cells were stained with 5-15 μg / ml class II dimers at room temperature for 4-5 h. After washing, cell surface molecules were counterstained with APC-Cy7-conjugated anti-CD4 mAb. + Absolute cell numbers were determined by flow cytometry.

[0190] DP4 dimer + T cell expansion and establishment of single T cell clones DP4 L112W / V141M dimer + For T cell proliferation, CD4 + T cells were stimulated as described above and DP4 L112W / V141M Stained with dimer. Dimer + Cells were sorted using anti-PE magnetic beads and expanded with artificial APC / mOKT3 cells irradiated at 200 Gy at E:T ratios of 5-20:1 (see Butler, MO et al., PLoS One 7, e30229 (2012)). Culture medium supplemented with IL-2 (10 IU / ml) and IL-15 (10 ng / ml) was replenished every 2-3 days. After 2-3 weeks, T cells were cultured at DP4 L112W / V141M Dimer staining was performed. DP4 L112W / V141M dimer + Single cell clones were generated by limiting dilution as previously described (Su, LF et al., Immunity 38, 373-383 (2013)). Briefly, memory CD4 + T cells were purified and treated with DP4 without dasatinib pretreatment. L112W / V141M Stained with dimer. Dimer + Cells were sorted and then stimulated with 5 μg / ml PHA-P and PBMCs from multiple allogeneic donors irradiated at 20 Gy in 96-well plates. After 1 week of stimulation with IL-2 (100 IU / ml) and IL-15 (10 ng / ml), the culture medium was replenished and supplemented. After 2 weeks, single cell clones were isolated, designated DP4. L112W / V141MStained with dimer.

[0191] ELISPOT assay Cytokine ELISPOT assays were performed as previously reported (see Yamashita, Y. et al., Nat Commun 8, 15244 (2017); Anczurowski, M. et al., Sci Rep 8, 4804 (2018)). The present disclosure relates, for example, to the following: [1] An HLA class II molecule comprising a DP beta chain, wherein the DP beta chain comprises an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO:1. [2] An HLA class II molecule comprising a DP beta chain, wherein the DP beta chain comprises a substitution mutation at an amino acid residue corresponding to position 112 of SEQ ID NO: 1, and the substitution mutation is due to an amino acid other than leucine. [3] The HLA class II molecule according to [1] or [2], wherein the DP beta chain further comprises an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1. [4] An HLA class II molecule comprising a DP beta chain, wherein the DP beta chain comprises an amino acid other than valine at the amino acid residue corresponding to position 141 of SEQ ID NO:1. [5] An HLA class II molecule comprising a DP beta chain, wherein the DP beta chain comprises a substitution mutation at an amino acid residue corresponding to position 141 of SEQ ID NO: 1, and the substitution mutation is due to an amino acid other than valine. [6] The HLA class II molecule according to [4] or [5], wherein the DP beta chain further comprises an amino acid other than leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1. [7] The DP beta chain is an HLA class II molecule described in any one of [1] to [6] above, which comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 3, 4, and 5. [8] The HLA class II molecule according to any one of [1] to [3], [6] and [7] above, wherein the amino acid other than leucine has a hydrophobic side chain. [9] The HLA class II molecule according to [8], wherein the amino acid other than leucine is selected from the group consisting of alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan.

[10] The HLA class II molecule according to any one of [1] to [3] and [6] to [9] above, wherein the amino acid other than leucine is tryptophan.

[11] The HLA class II molecule according to any one of [3] to

[10] above, wherein the amino acid other than valine has a hydrophobic side chain.

[12] The HLA class II molecule according to

[11] , wherein the amino acid other than valine is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[13] The HLA class II molecule according to any one of [3] to

[12] above, wherein the amino acid other than valine is methionine.

[14] The HLA according to any one of [1] to

[13] , wherein the DP beta chain contains tryptophan at the amino acid residue corresponding to position 112 of SEQ ID NO: 1 and methionine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1.

[15] The HLA class II molecule according to any one of [1] to

[14] above, wherein the DP beta chain comprises the amino acid sequence set forth in SEQ ID NO:3.

[16] The HLA class II molecule according to any one of [1] to

[15] above, wherein the DP beta chain comprises the amino acid sequence set forth in SEQ ID NO:4.

[17] The DP beta chains are DPB1*01, DPB1*02, DPB1*03, DPB1*04, DPB1*05, DPB1*06, DPB1*08, DPB1*09, DPB1*10, DPB1*100, DPB1*101, DPB1*102, DPB1*103, DPB1*104, DPB1*105, DPB1*106, DPB1*107, DPB1*108, DPB1*109, DPB1*11, DPB1*110, DPB1*111, DPB1*112, DPB1*113, DPB1*114, DPB1*115, DPB1*116, DPB1*11 7, 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*13 0, DPB1*131, DPB1*132, DPB1*133, DPB1*134, DPB1*135, DPB1*136, DPB1*137, DPB1*138, DPB1*139, DPB1*14, DPB1*140, DPB1*141, DPB1*142, DPB1*14 3, 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*15 6, 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*16 9, 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、D、 PB1*617、DPB1*618、DPB1*619、DPB1*62、DPB1*620、DPB1*621、DPB1*622、DPB1*623、DPB1*624、DPB1*625、DPB1*626、DPB1*627、DPB1*628、DPB1*629、DPB1*63、DPB1*630、DPB1*631、DPB1*632、DPB1*633、DPB1*634、DPB1*635、DPB1*636、DPB1*637、DPB1*638、DPB1*639、DPB1*64、DPB1*640、DPB1*641、DPB1*642、DPB1*643、DPB1*644、DPB1*645、DPB1*646、DPB1*647、DPB1*648、DPB1*649、DPB1*65、DPB1*650、DPB1*651、DPB1*652、DPB1*653、DPB1*654、DPB1*655、DPB1*656、DPB1*657、DPB1*658、DPB1*659、DPB1*66、DPB1*660、DPB1*661、DPB1*662、DPB1*663、DPB1*664、DPB1*665、DPB1*666、DPB1*667、DPB1*668、DPB1*669、DPB1*67、DPB1*670、DPB1*671、DPB1*672、DPB1*673、DPB1*674、DPB1*675、DPB1*676、DPB1*677、DPB1*678、DPB1*679、DPB1*68、DPB1*680、DPB1*681、DPB1*682、DPB1*683、DPB1*684、DPB1*685、DPB1*686、DPB1*687、DPB1*688、DPB1*689、DPB1*69、DPB1*690、DPB1*691、DPB1*692、DPB1*693、DPB1*694、DPB1*695、DPB1*696、DPB1*697、DPB1*698、DPB1*699、DPB1*70、DPB1*700、DPB1*701、DPB1*702、DPB1*703、DPB1*704、DPB1*705、DPB1*706、DPB1*707、DPB1*708、DPB1*709、DPB1*71、DPB1*710、DPB1*711、DPB1*712、DPB1*713、DPB1*714、DPB1*715、DPB1*716、DPB1*717、DPB1*718、DPB1*719、DPB1*72、DPB1*720、DPB1*721、DPB1*722、DPB1*723、DPB1*724、DPB1*725、DPB1*726、DPB1*727、DPB1*728、DPB1*729、DPB1*73、DPB1*730、DPB1*731、DPB1*732、DPB1*733、DPB1*734、DPB1*735、DPB1*736、DPB1*737、DPB1*738、DPB1*739、DPB1*74、DPB1*740、DPB1*741、DPB1*742、DPB1*743、DPB1*744、DPB1*745、DPB1*746、DPB1*747、DPB1*748、DPB1*749、DPB1*75、DPB1*750、DPB1*751、DPB1*752、DPB1*753、DPB1*754、DPB1*755、DPB1*756、DPB1*757、DPB1*758、DPB1*759、DPB1*76、DPB1*760、DPB1*761、DPB1*762、DPB1*763、DPB1*764、DPB1*765、DPB1*766、DPB1*767、DPB1*768、DPB1*769、DPB1*77、DPB1*770、DPB1*771、DPB1*772、DPB1*773、DPB1*774、DPB1*775、DPB1*776、DPB1*777、DPB1*778、DPB1*779、DPB1*78、DPB1*780、DPB1*781、DPB1*782、DPB1*783、DPB1*784、DPB1*785、DPB1*786、DPB1*787、DPB1*788、DPB1*789、DPB1*79、DPB1*790、DPB1*791、DPB1*792、DPB1*794、DPB1*795、DPB1*796、DPB1*797、DPB1*798、DPB1*799、DPB1*80、DPB1*800、DPB1*801、DPB1*802、DPB1*803、DPB1*804、DPB1*805、DPB1*806、DPB1*807、DPB1*808、DPB1*809、DPB1*81、DPB1*810、DPB1*811、DPB1*812、DPB1*813、DPB1*814、DPB1*815、DPB1*816、DPB1*817、DPB1*818、DPB1*819、DPB1*82、DPB1*820、DPB1*821、DPB1*822、DPB1*823、DPB1*824、DPB1*825、DPB1*826、DPB1*827、DPB1*828、DPB1*829、DPB1*83、DPB1*830、DPB1*831、DPB1*832、DPB1*833、DPB1*834、DPB1*835、DPB1*836、DPB1*837、DPB1*838、DPB1*839、DPB1*84、DPB1*840、DPB1*841、DPB1*842、DPB1*843、DPB1*844、DPB1*845、DPB1*846、DPB1*847、DPB1*848、DPB1*849、DPB1*85、DPB1*850、DPB1*851、DPB1*852、DPB1*853、DPB1*854、DPB1*855、DPB1*856、DPB1*857、DPB1*858、DPB1*859、DPB1*86、DPB1*860、DPB1*861、DPB1*862、DPB1*863、DPB1*864、DPB1*865、DPB1*866、DPB1*867、DPB1*868、DPB1*869、DPB1*87、DPB1*870、DPB1*871、DPB1*872、DPB1*873、DPB1*874、DPB1*875、DPB1*876、DPB1*877、DPB1*878、DPB1*879、DPB1*88、DPB1*880、DPB1*881、DPB1*882、DPB1*883、DPB1*884、DPB1*885、DPB1*886、DPB1*887、DPB1*888、DPB1*889、DPB1*89、DPB1*890、DPB1*891、DPB1*892、DPB1*893、DPB1*894、DPB1*895、DPB1*896、DPB1*897、DPB1*898、DPB1*899、DPB1*90、DPB1*900、DPB1*901、DPB1*902、DPB1*903、DPB1*904、DPB1*905、DPB1*906、DPB1*907、DPB1*908、DPB1*909、DPB1*91、DPB1*910、DPB1*911、DPB1*912、DPB1*913、DPB1*914、DPB1*915、DPB1*916、DPB1*917、DPB1*918、DPB1*919、DPB1*92、DPB1*920、DPB1*921、DPB1*922、DPB1*923, DPB1*924, DPB1*925, DPB1*926, DPB1*927, DPB1*928, DPB1*929, DPB1*93, DPB1*930, DPB1*931, DPB1*932, DPB1*933, DPB1*934, DPB1* 935, DPB1*936, DPB1*937, DPB1*938, DPB1*939, DPB1*94, DPB1*940, DPB1*941, DPB1*942, DPB1*943, DPB1*944, DPB1*945, DPB1*946, DPB1*947, D The HLA class II molecule according to any one of [1] to

[16] above, which is selected from DPB1*948, DPB1*949, DPB1*95, DPB1*950, DPB1*951, DPB1*952, DPB1*953, DPB1*954, DPB1*955, DPB1*956, DPB1*957, DPB1*958, DPB1*959, DPB1*96, DPB1*960, DPB1*961, DPB1*962, DPB1*963, DPB1*964, DPB1*965, DPB1*97, DPB1*98, and DPB1*99.

[18] The HLA class II molecule according to any one of [1] to

[17] above, wherein the DP beta chain comprises the amino acid sequence set forth in SEQ ID NO:1.

[19] The HLA class II molecule according to any one of [1] to

[18] above, further comprising a DP alpha chain.

[20] The aforementioned DP alpha chain is DPA1*01:03:01:01, DPA1*01:03:01:02, DPA1*01:03:01:03, DPA1*01:03:01:04, DPA1*01:03:01:05, DPA1*01:03:01:06, DPA1*01:03:01:07, DPA1*01:03:01:08, DPA1*01:03:01:09, DPA1*01:03:01:10, DPA1*01:03:01:11, DPA1*01:03:01:12, DPA1*01:03:01:13, DPA1*01:03:01:14, DPA1*01:03:01:15, DPA1*01:03:01:16, DPA1*01:03:01:17, DPA1*01:03:01:18Q, DPA1*01:03:01:19, DPA1*01:03:01:20, DPA1*01:03:01:21, DPA1*01:03:01:22, DPA1*01:03:01:23, DPA1*01:03:02, DPA1*01:03:03, DPA1*01:03:04, DPA1*01:03:05, DPA1*01:03:06, DPA1*01:03:07, DPA1*01:03:08, DPA1*01:03:09, DPA1*01:04, DPA1*01:05, DPA1*01:06:01, DPA1*01:06:02, DPA1*01:07, DPA1*01:08, DPA1*01:09, DPA1*01:10, DPA1*01:11, DPA1*01:12, DPA1*01:13, DPA1*01:14, DPA1*01:15, DPA1*01:16, DPA1*01:17, DPA1*01:18, DPA1*01:19, DPA1*02:01:01:01, DPA1*02:01:01:02, DPA1*02:01:01:03, DPA1*02:01:01:04, DPA1*02:01:01:05, DPA1*02:01:01:06, DPA1*02:01:01:07, DPA1*02:01:01:08, DPA1*02:01:01:09, DPA1*02:01:01:10, DPA1*02:01:01:11, DPA1*02:01:02:01, DPA1*02:01:02:02, DPA1*02:01:03, DPA1*02:01:04, DPA1*02:01:05, DPA1*02:01:06, DPA1*02:01:07,DPA1*02:01:08:01, DPA1*02:01:08:02, DPA1*02:02:02:01, DPA1*02:02:02:02, DPA1*0 2:02:02:03, DPA1*02:02:02:04, DPA1*02:02:02:05, DPA1*02:02:03, DPA1*02:02:04, D PA1*02:02:05, DPA1*02:02:06, DPA1*02:03, DPA1*02:04, DPA1*02:05, DPA1*02:06, DPA 1*02:07:01:01, DPA1*02:07:01:02, DPA1*02:07:01:03, DPA1*02:08, DPA1*02:09, DPA1* 02:10, DPA1*02:11, DPA1*02:12, DPA1*02:13N, DPA1*02:14, DPA1*02:15, DPA1*02:16, D PA1*03:01:01:01, DPA1*03:01:01:02, DPA1*03:01:01:03, DPA1*03:01:01:04, DPA1*03 The HLA class II molecule according to

[19] , which is selected from 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, and DPA1*04:01:01:03, DPA1*04:02. [twenty one] The HLA class II molecule of

[19] or

[20] , wherein the DP alpha chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 6 or 8. [twenty two] The HLA class II molecule according to

[21] , wherein the DP alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 6 or 8. [twenty three] The HLA class II molecule according to any one of [1] to

[22] above, which is a DP1, DP2, DP3, DP4, DP5, DP6, DP8, or DP9 allele. [twenty four] The HLA class II molecule according to any one of [1] to

[23] , wherein the DP beta chain has a higher affinity for CD4 protein compared to a reference HLA class II molecule, and the reference HLA class II molecule comprises a DP beta chain containing (i) a leucine at the amino acid residue corresponding to position 112 of SEQ ID NO: 1, and / or (ii) a valine at the amino acid residue corresponding to position 141 of SEQ ID NO: 1. [twenty five] The HLA class II molecule according to

[24] , wherein the affinity 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, or at least about 1000 times higher.

[26] The HLA class II molecule according to any one of [1] to

[25] above, wherein the DP beta chain is bound to a cell membrane.

[27] The HLA class II molecule according to any one of [1] to

[25] above, wherein the DP beta chain is not bound to a cell membrane.

[28] The HLA class II molecule according to any one of [1] to

[25] above, wherein the DP beta chain comprises the extracellular domain of a full-length DP alpha chain.

[29] The HLA class II molecule according to

[28] , wherein the DP beta chain does not include the transmembrane domain of a full-length DP beta chain.

[30] The HLA class II molecule according to any one of

[19] to

[29] above, wherein the DP alpha chain is bound to a cell membrane.

[31] The HLA class II molecule according to any one of

[19] to

[29] above, wherein the DP alpha chain is not bound to a cell membrane.

[32] The HLA class II molecule according to any one of

[19] to

[29] above, wherein the DP alpha chain comprises the extracellular domain of a full-length DP alpha chain.

[33] 32. An HLA class II molecule according to claim 32, wherein the DP alpha chain does not contain the transmembrane domain of a full-length DP alpha chain.

[34] The HLA class II molecule according to any one of

[27] to

[33] above, wherein the DP beta chain is linked or bound to an inert particle.

[35] The HLA class II molecule according to

[34] , wherein the inert particles are beads.

[36] The HLA class II molecule according to

[35] , wherein the inert particles are nanoparticles.

[37] 36. The HLA class II molecule according to claim 35, wherein the nanoparticles are selected from PEGylated iron oxide, chitosan, dextran, gelatin, alginate, liposomes, starch, branched polymers, carbon-based carriers, polylactic acid, poly(cyano)acrylate, polyethyleinemine, block copolymers, polycaprolactone, SPIONS, USPIONS, Cd / Zn-selenide, or silica nanoparticles.

[38] The HLA class II molecule according to

[36] or

[37] , wherein the nanoparticles are PEGylated iron oxide nanoparticles.

[39] The HLA class II molecule according to any one of [1] to

[38] above, wherein the DP beta chain comprises a signal peptide.

[40] The HLA class II molecule according to any one of

[19] to

[39] above, wherein the DP alpha chain comprises a signal peptide.

[41] The HLA class II molecule according to

[39] or

[40] , wherein the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:9.

[42] A nucleic acid molecule encoding the DP beta chain according to any one of [1] to

[41] above.

[43] The nucleic acid molecule according to

[42] above, further encoding the DP alpha chain according to any one of

[19] to

[41] above.

[44] The nucleic acid molecule according to

[42] or

[43] , comprising a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:2.

[45] A vector comprising the nucleic acid molecule according to any one of

[42] to

[44] above.

[46] A cell comprising the HLA class II molecule according to any one of [1] to

[41] above, the nucleic acid molecule according to any one of

[42] to

[44] above, or the vector according to

[45] above.

[47] The cell according to

[46] above, which is a mammalian cell or an insect cell.

[48] The cell according to

[46] or

[47] , which is selected from K562 cells, T2, HEK293, HEK293T, A375, SK-MEL-28, Me275, COS, fibroblasts, tumor cells, or any combination thereof.

[49] The cell according to any one of

[46] to

[48] above, which lacks endogenous MHC class II DP beta chain expression.

[50] The cell according to any one of

[46] to

[49] above, which lacks endogenous MHC class II DP alpha chain expression.

[51] A method for identifying a T cell receptor capable of binding to an epitope of an MHC class II complex, comprising pulsing the cell according to any one of

[46] to

[50] above with one or more peptides containing the epitope, and activating one or more CD4 + stimulating the T cells with APCs.

[52] A method for treating a disease or pathological condition in a subject in need thereof, the method comprising administering to the subject an MHC class II molecule described in any one of [1] to

[41] above.

[53] The method according to

[52] above, wherein the disease or condition is cancer or infection.

[54] The method of

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

[55] The method according to

[53] or

[54] , wherein the cancer is recurrent or refractory.

[56] The method according to any one of

[53] to

[55] above, wherein the cancer is locally progressive.

[57] The method according to any one of

[53] to

[56] above, wherein the cancer is progressive.

[58] The method according to any one of

[53] to

[57] above, wherein the cancer is metastatic.

[59] K less than approximately 10 μM D The HLA class II molecule according to any one of [1] to

[41] above, which binds to CD4 at the

[60] K below approximately 8.9 μMD The HLA class II molecule according to any one of [1] to [1] above, which binds to CD4 at the

[61] A complex comprising an HLA class II molecule according to any one of [1] to

[41] ,

[59] and

[60] above, and a peptide, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 to 237.

Claims

1. An HLA class II molecule comprising an extracellular domain of a DP beta chain comprising the amino acid sequence set forth in SEQ ID NO:

3.

2. The HLA class II molecule of claim 1, wherein the extracellular domain of the DP beta chain comprises the amino acid sequence set forth in SEQ ID NO:

4.

3. An HLA class II molecule as described in claim 1, wherein the extracellular domain of the DP beta chain comprises the amino acid sequence set forth in SEQ ID NO:

240.

4. The HLA class II molecule of any one of claims 1 to 3, further comprising a DP alpha chain.

5. The HLA class II molecule of claim 4, wherein the DP alpha chain comprises the amino acid sequence set forth in SEQ ID NO:

6.

6. The HLA class II molecule of claim 4, wherein the DP alpha chain comprises the amino acid sequence set forth in SEQ ID NO:

8.

7. The HLA class II molecule according to any one of claims 1 to 6, wherein the DP beta chain is bound to a cell membrane.

8. The HLA class II molecule of any one of claims 1 to 6, wherein the DP beta chain is linked or bound to an inert particle.

9. The HLA class II molecule of any one of claims 4 to 8, wherein the DP alpha chain comprises a signal peptide.

10. An HLA class II molecule described in any one of claims 4 to 9, wherein the DP alpha chain is bound to a cell membrane.

11. An HLA class II molecule described in any one of claims 4 to 10, wherein the DP alpha chain comprises the extracellular domain of a full-length DP alpha chain.

12. A complex comprising an HLA class II molecule according to any one of claims 1 to 11 and a peptide, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 32 to 237.

Images