用于高级 NK 细胞免疫疗法的多重 iPSC 平台Multiplexed iPSC platform for advanced NK cell immunotherapies

Pleasecitethisarticleinpressas:Kumaretal.,MultiplexediPSCplatformforadvancedNKcellimmunotherapies,CellReportsMedicine(2025),

/10.1016/j.xcrm.2025.102282

cellReportsMedicine

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Review

MultiplexediPSCplatformforadvancedNKcellimmunotherapies

AkhileshKumar,

1

ColinFischer,

2

FrankCichocki,

1

andJeffreyS.Miller

1,2,

*

1DivisionofHematology,OncologyandTransplantation,UniversityofMinnesota,Minneapolis,MN,USA

2MasonicCancerCenter,UniversityofMinnesota,Minneapolis,MN,USA

*Correspondence:

mille011@

/10.1016/j.xcrm.2025.102282

SUMMARY

Humanpluripotentstemcell(PSC)derivationadvanceshaverevealedenormouspotentialforimprovedcancerimmunotherapyandclinical-scalebloodcellproduction.PSCscanself-renewindefinitelyandbedifferentiatedintospecializedcells,makingthempromisingcandidatesforproducingcytotoxiclympho-cytes.Derivingnaturalkiller(NK)cellsfromPSCsunlocksnewpossibilitiesforstudyingdevelopmentalhematopoiesisandinvestigatingpotentialimmunotherapytreatments.Cellulartherapies,combinedwithge-neticengineering,arepotenttoolsforcombatingcancerandviralinfections.WhileNKcellsdirectlylysetu-morcells,geneticmodifications,suchaschimericantigenreceptor(CAR)engineeringorthedeletionofcheckpointmolecules,canenhancetheirfunctionalcapacity.Here,wediscussrecentadvancesininducedPSC(iPSC)editingandguideddifferentiation,focusingondevelopingNKcellimmunotherapeuticproductsandoptimizingiPSCsasanNKcellsourcetobroadentherapeuticoptionsandaddressdiversepatientneeds.ThiscomprehensivereviewevaluatesiPSC-derivedNKcell-basedtherapies,recentadvances,andfuturegenome-editingstrategies.

INTRODUCTION

Progressincellularimmunotherapyhastransformedthethera-peuticlandscapeforcancerandotherailments.Overthelastdecade,numerouscellulartherapieshaveadvanced,frompre-clinicalstudiestoFoodandDrugAdministration(FDA)-approvedtreatments.Recentdiscoveriesinimmunology,geneticengi-neering,andcellproductionhavefacilitatednoveltherapeuticstrategies.Liveimmunecellinterventionspresentthepossibilityofcurativeresultsincancersandchronicinfectionsthatareun-responsivetoconventionalclinicallyapprovedtreatments.Livingcellsoffersignificantadvantagesduetotheirabilitytoadapttoenvironmentalchangesandparticipateincomplexsignalingpathwaysthattraditionaldrugscannot.

1

Amongthese,chimericantigenreceptor(CAR)-Tcelltherapyhasgarneredattentionasagroundbreakingapproachintreatingcertainhe-matologicalcancers,withseveralshowingremarkablesuccessinclinicaltrialsandreceivingFDAapproval.

2

Despitethesepromisingoutcomes,CAR-Tcelltherapyfacessignificantchal-lenges.Currentclinicallyusedtreatmentsareexpensive,requirecomplexmanufacturing,andcancauseserioussideeffectssuchascytokinereleasesyndrome(CRS)andneurotoxicity.Forcon-ditionslikeBcellacutelymphoblasticleukemia,Bcelllym-phomas,andmultiplemyeloma(MM),addressingtheseissuesremainscriticaltoexpandingthepotentialofCAR-Tcelltreat-ments.

2

,3

Thesehurdleshaveledresearcherstoexplorealterna-tiveimmunecelltherapies.Naturalkiller(NK)cellsarepartoftheinnateimmunesystemandcanemployvariouskillingmodalitieswithoutantigen-specificrecognition.Thisallowsthemtotargeta

broadrangeofcancercellswithoutcomplexengineeringorchal-lengingengraftmentrequirements,makingthemarelativelysafertherapeuticapproachthantheirTcellcounterparts.

4

,5

Withrecentadvancementsinreprogrammingtechnologies,inducedpluripotentstemcells(iPSCs)haverevolutionizedthefieldofcellularimmunotherapy.

6–8

Thesedevelopments,builtoverdecades,arenowbeingappliedtotreatseveraldiseasesaffectingvarioustissues,includingskin,heart,muscle,andblood.

9

,

10

iPSCsprovidearenewableanduniversalsourceforimmunecellgenerationwithconsistentqualityandscalability,makingthem‘‘off-the-shelf’’(OTS)cellulardrugs.TargetedimmunotherapyhasadvancedsignificantlywiththeintroductionofNKcellsderivedfromiPSCs,particularlywiththeuseofCARtechnology.Thisinnovationsubstantiallybroadensthethera-peuticlandscapeforNKcells.CAR-NKcellshavethepotentialtorevolutionizecancertreatment,buttheirpotentialapplicationsinfibroticdiseases,endometriosis,andneurologicaldisordersarejustbeginningtobeexplored.Inthisreview,wediscussthecurrentadvancementsandchallengesfacedinthedenovogenerationofNKcellsfromiPSCs(iPSC-NK),aswellashowiPSCtechnologiescanassistinovercomingmajorobstaclesthatpresentlyhindercellularimmunotherapies.

NKCELLS:ASYMPHONYOFIMMUNITY

NKcellsoriginatefromcommonlymphoidprogenitors(CLPs)thatarederivedfromhematopoieticstemcells(HSCs)inthebonemarrow(BM)andareintegraltoinnateimmunity.

11

,

12

NKcellsaredistinguishedfromotherlymphoidcellsbytheabsence

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/licenses/by-nc-nd/4.0/

).

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Co-Activ

A

ti

CD16

NKP80on

NKp46

<

NKP44

CD56dimNKCells

CD56brightNKCells

NKCell

NKP30

peripheralBlood

secondaryLymphoid

人

Tissues

15

Macrophages,DCs,BandTCells

stressed,Malignant,Infectedcells

LinearModelofHumanNKCellDevelopment

B

HSCMPPCLPNKPCD56brightNKCD56dimNK

Lin-

CD34+

CD38'OW-CD45RA-

CD122+NKG2D+

NKP46+

NKP44+

NKP30+CD16

KIR-

CD57

Lin-

CD122+

CD38+

NKG2D+CD45RA+CD7+

CD123-

Lin-

CD34+

CD38-

CD45RA+

CD90+

Lin-

CD34low

CD38+

CD45RA+

CD122+NKG2D+

NKG2A-NKP46+

NKP44+

NKP30+CD16+KIR+

CD57+

Non-LinearModelofHumanNKCellDevelopment

AdaptiveNK

CD56dimNKG2A-KIR+

CD57+

NKG2chiPLZF-

CMV-

CLPNKPCD56brightNK

MPP

HSC

Lin,CD122+,

NKG2D+,

CD45RA+,CD7+

Lin,CD34low,

CD38+,CD45RA+

CD122+,NKG2D+,

NKP46+,NKP44+,

NKP30+,CD16,KIR-

Lin

CD34+

CD38'OW-CD45RA-

Lin-

CD34+

CD38

CMP

Lin,CD34bw,

CD33+,CD45RA+

NKP

Lin,CD122+,

NKG2D+,

CD45RA+,CD7+

CD56dimNK

CD122+,NKG2D+,

NKP46+,NKP44+,NKP30+,

CD16,KIR+,CD57+

CD45RA+CD90+-

AdaptiveNK

CD56dimNKG2A-KIR+

CD57+

NKG2chiPLZF-

CMV-

Figure1.MajorrolesanddevelopmentofNKcellsinhumans

(A)NKcellsreleasecytotoxicproteinsinresponsetointeractionwithstressed,malignant,orinfectedcells.NKcellscanmodulateadaptiveimmunecellabundance,maturity,andactivitybysecretionofIFN-γandTNF-α.CD56dimNKcellspredominateperipheralbloodNKcellpopulationsandprimarilyperformcytolyticactivityandimmunesurveillance.IL-15secretedbymultiplecelltypescanimpactNKcellactivitythroughRas/Raf,PI3K,andJAK/STATpathways.CD56brightNKcellsmakeupmostNKcellsresidinginsecondarylymphoidtissues,wheretheyperformprimarilyimmune-modulatingfunctions.

(legendcontinuedonnextpage)

2CellReportsMedicine6,102282,October21,2025

(B)Linearandnon-linearmodelsofhumanNKcelldevelopment.Inthelinearmodel,hematopoieticstemcells(HSCs)generatelymphoid-primedmultipotentprogenitors(MPPs),whichdifferentiateintocommonlymphoidprogenitors(CLPs)andthenNKcellprecursors(NKPs).TheseprecursorsmaturesequentiallyintoCD56brightandCD56dimNKcells,withadaptiveNKcellsemerginglaterinresponsetoviralinfection.Thenon-linearmodelsuggestsamoreflexibledevelop-mentaltrajectory.HSCsstillgenerateMPPs,butthesecandifferentiateintoeitherCLPsorcommonmyeloidprogenitors(CMPs),bothcapableofproducingNKPs.CLPspredominantlygiverisetoCD56brightNKcells,whileCMPstendtogenerateCD56dimNKcells,bothofwhichcanultimatelydifferentiateintoadaptiveNKcells.ThismodelunderscoresthegrowingrecognitionofNKcellheterogeneityanddevelopmentalplasticityindifferentphysiologicalanddiseasesettings.

CellReportsMedicine6,102282,October21,20253

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ofCD3andhighlevelsofCD56expression,andtheirsurvivalandproliferationaredrivenbyinterleukin(IL)-15signalingthroughtheJAK/STAT,phosphatidylinositol3-kinase(PI3K),andRas/Raf

pathways.13

Uponmaturation,NKcellscanmigratefromtheBMtothebloodstreamandestablishthemselvesinnumeroustissuesduetotheirabilitytomovebetweenlymphaticandnon-lymphoidorgans.

12

,14

,15

FullymatureNKcellsacquireeffectorroles,includingnaturalcytotoxicityagainststressed,malignant,andvirallyinfectedcells,aswellasthemodulationofadaptiveimmuneresponsesthroughthesecretionofcyto-kines,chemokines,andgrowthfactors

16

,17

(

Figure1

).IncontrasttoTcells,NKcellscanrapidlyrecognizeandkilltargetcellswithoutpriorsensitization.

11

Developmentalinsight

UnderstandingthedevelopmentofhumanNKcellsandotherinnatelymphoidcell(ILC)lineagesiskeytothedevelopmentofcellularproducts.However,canonicalNKcelldevelopmentisinsufficientlyunderstood,creatingchallengesforinvitroreplica-tion.Thecurrentpredominantmodelproposesalinear,gradualtransitionwhereCLPsdecreaseCD34expressionandincreaseCD56expression.

18

,19

TheearlieststagesofNKcelldevelopmentinthetonsilbeginwithCD34+CD38−CD45RA+CD10+CD7+CD117−CD94−CD16−CLPspossessingPro-B,Pre-T,andNKprogenitor(NKP)potential,aswellaspotentialforotherILCline-ages

.18

,20

,21

TheacquisitionofIL-1R1marksthebeginningofNKP(Lin−IL1R1+CD122+CD38+CD123−CD45RA+CD7+)commit-ment.NKcelllineagespecificationfromCLPs,whichcanalsobefoundincirculation,impliesthatCD122+NKPcellsareadevelop-mentalintermediatecapableofseedingperipheraltissuesbeforefurtherdifferentiation

.12

,14

NKPsthendifferentiateintomorerestrictedimmatureNK(iNK)precursorcells,characterizedbyhigherexpressionofIL-1R1andtheappearanceofCD314(NKG2D),CD335(NKp46),CD337(NKp30),andCD161(KLRB1).ThenexttransitionismarkedbytheemergenceofCD117+/−CD94+CD16−CD56brightNKcells,characterizedbyelevatedCD56levelsalongsidemaximalexpressionofNKG2D,NKp46,NKp30,andKLRB1.Subsequently,theseCD56brightNKcellsfurtherdifferentiateintoCD117−CD94+/−CD16+CD56dimNKcells(CD56dimNK),whicharedistinguishedbyreducedCD56levels,heightenedCD16expression,andtheacquisitionofkillerimmunoglobulin-likereceptors(KIRs)

12

,18

,20

(

Figure1

A).Insupportofalineardevelopmentmodel,CD56brightNKcellsdisplaylongertelomeresthanCD56dimNKcellsandcanacquireCD16andKIRuponactivation.Furthermore,studiesusingadop-tivetransferintomicedemonstratedtheapparentmaturationofCD56brightcells

.22–24

Recently,anon-linearmodelofhumanNKcelldevelopmentwasproposed,assertingthatcommonmyeloidprogenitorsandgran-

ulocyte-monocyteprogenitorscandifferentiateintoNKcellswhenexposedtoNK-supportingcytokinesandstromalcells

20

,25

(

Figure1

B).Furthermore,researchsuggeststhatCD56brightandCD56dimNKcellsmayhavedifferentontog-enies

.20

,26

CD56brightNKcellsinperipheralbloodexhibitlowcytotoxiccapacitybutdisplaystrongcytokinesecretionwhenstimulated.Conversely,CD56dimNKcellshaverobustnaturalcytotoxicity,canexecuteantibody-dependentcellularcytotoxicity(ADCC),anddisplayanabundanceofcytolyticgranules.

22

,23

,27

Ev-idenceshowsthattheCD56dimNKcellpopulationcanalsoserveasareservoirforNKcellspossessingimmunologicalmemoryforencounterswithhaptens,cytomegalovirus,orinflammatorycyto-kinestimulation.Memory(ormemory-like)NKcellsarephenotyp-icallydistinctsubsetscharacterizedbyenhancedpersistence,metabolicfitness,andantitumoractivity.Theyalsoexhibitspecificandamplifiedresponsestosecondaryexposureevents

.19

,25

,28

ContrastinghallmarksofNKcellsvs.Tcells

ConsideringthepivotalroleofNKcellresponsesintumorimmunity,promisingavenuesexistforharnessingthisactivityinimmunotherapy.NKcellspossessabroaddistributionofacti-vatingreceptorsforrecognizingdistressedandtransformedcellsandcanquicklydistinguishandlysetumorcellswithoutantigenpriming.

29

,

30

Thisnaturalcytotoxicitycancomplementantigen-specifictargetingapproachesforNKcelltherapy.Evenwhenidealtumortargetsareavailable,antigenescapemechanismspresentsignificanthurdlestosuccessfulTcellimmunotherapy.

30

,31

Forinstance,humanleukocyteantigen(HLA)-Idownregulationisacommontacticexploitedbymalig-nantcellstoevadeTcellreceptor(TCR)-mediatedimmunity,im-pactingmorethan90%ofpatientsinsomecancertypes.NKcellsreadilyidentifyanddestroyinfectedortransformedcellslackingHLA-Ithroughaphenomenonknownas‘‘missingself.’’

32

,33

WhileTcelltreatmentshavekeyadvantages,suchasenhancedmemoryresponseandprolongedinvivosurvival,theuniqueattributesofNKcellbiologyrenderthempromisingcandidatesindiseaseswhereTcellantitumoractionisinsuffi-cientorintolerable.Althoughprimarilyactiveinearlytumorcontrol,NKcellsalsoplayaroleinlate-stagedisease,demon-stratingthecapacitytolimitmetastasisthroughinterferon(IFN)-γproductionordirectcytotoxicity.

34

,35

Antigenescapeoftenaccompaniesmetastasisasafunctionoftumorevolution,contributingtopoorTcellactivityagainstre-sidualandmetastaticdisease.A‘‘cold’’tumorpossessinganimmunosuppressivetumormicroenvironment(TME)isanaddi-tionalnegativeprognosticmarkerofTcell-basedtherapies.

36

,37

Throughtheirmodulationofadaptiveimmunity,NKcellsmaybecapableofturningcoldtumors‘‘hot’’byenhancingmacrophageanddendriticcellantigenpresentationandstimulatingTandB

4CellReportsMedicine6,102282,October21,2025

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Table1.AcomparativeanalysisofNKcellsandTcellsthroughdefiningfeaturesoftumorimmunity

Killingmodalities•contact-dependentdeathligand-medi-

atedlysis

•ADCC

•recognitionof‘‘lackofself’’

•recognitionofstressligands

CharacteristicsNKcellsTcells

•TCR-mediatedlysis,highlyspecific

•contact-dependentdeathligand-medi-atedlysis

Memorypotential•minimal,containedwithinsmallsub-

populations(CIMLandadapt-NK)

•highandrobust,butsmallfractionofto-talpopulations

Immunemodulation•extensive,keyregulatorsofadaptive

immunity

•moderate,releaseimmune-stimulatingcytokinesuponkilling

Immunosuppression

•

•

•

•

activatedbyMHCdownregulation

inhibitedbyTregcells

inhibitedbyTGF-β

inhibitedbyimmunecheckpointproteins

•

•

•

•

heavilyimpactedbyMHCdownregula-tion

inhibitedbyTregcells

inhibitedbyTGF-β

inhibitedbyimmunecheckpointproteins

Researchfeasibility

•

•

•

•

lowyieldsfromdonorblood

moderatelyexpandable

lowviralandplasmid-basedgenetrans-

ferefficiency

sensitiveculturerequirements(bothpri-

maryNKcellsandiPSC-derivedNK

cells)

•

•

•

•

highyieldsfromdonorblood

highlyexpandable

highfeasibilityofgenetransferrelativelyinsensitiveculture

Clinicaluse•positivesafetyprofile

•fewengraftments’requirements

•potentialbenefitsoutsideofcancer:HIVandmalaria

•fewFDA-approvedtherapies

•moderatesafetyprofile;commonoff-tu-mortoxicities

•numerousengraftmentrequirementstoavoidtoxicities

•multipledirectcelltherapiesandauxiliarydrugsareFDAapproved

cellantitumoractivity.

38

,39

Tcelltherapiescanresultinhighinci-dencesofadverseeventssuchasCRS,neurotoxicity,andgraft-vs.-hostdiseaseduetooff-tumoreffects.

40

,41

Incontrast,NKcellinfusionsposeminimalrisksofthesecomplications,evenwithallogeneicgrafts.

42

,

43

Together,thesepositiveimpactsofNKcellsonimmuneregulationanddiseasecontrolsupporttheircontinueduseasindependentcellulartherapiesorinconjunc-tionwithothercancertreatments(

Table1

).

Guardiansofhealthandfightersofdisease

Aspartoftheinnateimmunesystem,NKcellsarecrucialincombatingcancer,infectiousdiseases,autoimmunedisorders,andchronic

inflammation.30

,44

NKcellsactasafirstlineofde-fenseagainstinfectedandabnormalcellsbymonitoringcellstressandactivelydetectinghematologicalandsolidcancers,circulatingtumorcells,andmetastatic

progression.17

,45

,46

Notably,cytotoxicNKcellinfiltrationintotumorsisafavorableprognosticindicatorformelanoma,renalcellcarcinoma,andliver,lung,andbreastcancer

.47–49

NKcellkillingisinitiatedwhenthebalanceofinhibitoryandactivatingsignalsreceivedbytheNKcellistippedbypro-inflammatorycytokines,engagementofacti-vatingreceptorsisincreased,orthereisalossofengagementthroughinhibitoryreceptors.

16

NKcellkillingencouragesfurtherinflammationthroughcytokinesecretion,whichstimulateseffectorimmunepopulationstoenhancetheimmuneresponse.NKcellsplayaroleinbothearly-andlate-stagemalignancies.However,immunosuppressivefactorsintheTMEcandetertheirimpactinboth,includingtransforminggrowthfactorβ(TGF-β)

andregulatorycellssuchasTregs,anti-inflammatorymacro-phages,andN2-likeneutrophils.

50

NKcellsalsopossessnumerousregulatorymechanismsexploitablebytumors,includingcheckpointreceptors,NKcelllocalizationfactors,andNKcellself-tolerancemechanisms.Noveltherapeuticstrategiesmustaccountforthesefeaturestoavoidlimitedtreatmentre-sponses.Whenthistolerancefailsinhealthyindividuals,NKcellscanparticipateintheemergenceofnumerousautoimmunedis-eases,wheretheyhavebeendescribedasprotectiveand

pathogenic.51

NKcellcytokineproductioncanactivateimmaturedendriticcells,macrophages,andCD4+TcellsviaIFN-γandtu-mornecrosisfactor(TNF)secretion.Reciprocalinteractionsbe-tweenNKandotherimmunecellscanmodulateoractivateNKcellproliferationand

cytotoxicity.16

NKcellsarealsophysicallylocalizedtooptimizetheirimmunefunctions,withmorecytotoxicCD56dimpopulationsservingassentrycellsintheperipheralbloodandmorecytokine-producingCD56brightpopulationslocal-izingtolymphoidtissuewheretheycaninfluenceotherimmunecells

.15

,16

,52

ThefunctionsofNKcellsinhealthytissues,particu-larlyinsecondarylymphoidtissues,arebecomingatopicofincreasinginvestigation,andeffortsareongoingtotranslateNKcellfunctionsinhealthytissues

intotherapeuticadvances.53

HARNESSINGNKCELLS:THEPROMISEOFADOPTIVEIMMUNOTHERAPY

Immunotherapyhasrapidlytransformedclinicaloncology,ce-mentingitselfasthefourtharmofcancertherapyinadditionto

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surgery,radiotherapy,andchemotherapy.InafielddominatedbyTcellstudies,NKcellsaregainingtractionduetotheirbene-ficialsafetyprofiles,immunoregulatoryfunctions,andcapacityforquick,effectivetargetingandkillingoftumorcells.

54

,55

Ther-apeuticNKcellscanbegatheredfrommultipleprimaryandsyn-theticsources,eachwithuniqueimplicationsfortheirclinicaluse.ManyapproachesuseNKcellscollectedfromhealthydonorperipheralblood.Despitecomprisingasmallfractionoftotallymphocytes(~5%),peripheralbloodNKcellscanbeen-richedandexpandedwithengineeredfeedercellstoobtainlargenumbersforimmunotherapy.

56

,57

Alternatively,NKcellscanbeharvestedfromumbilicalcordbloodunitsandexpandedtoclin-icallyrelevantlevels.

56

Despiteoriginatingfromdifferenttissues,peripheralandcordbloodNKcellsdemonstratesimilarrelativecytotoxicityagainsttumorcells.

58

,59

Whileefficacious,primary-sourcedNKcellyieldsarehighlyinconsistentbetweendonorsandareheavilyinfluencedbybatchpurificationmethods.

30

,60

NaivecordbloodNKcellsalsoexhibitarelativelyimmaturephenotype,withlowexpressionofCD16,KIRs,andkeyNK

celladhesionmoleculessuchasCD2.61

However,thislimitationcanbeovercomethroughexvivoexpansionintofunctionallymatureNKcells,asdemonstratedbyLuevanoetal.,usingcyto-kine-baseddifferentiationprotocols.

62

Onegrouprecentlydescribedapotentialthirdsourceofdonor-derivedNKcells,isolatedandexpandedfromfull-termhumanplacentas,offeringanadditionalpromisingalternativeforNKcell-basedimmunotherapies.

63

NKcelllineshavebeenestablishedforindefiniteculturetoavoiddonorvariationandfacilitateconsistentyield,purification,andcellcharacteristics,

64

buttherearechallenges.OneclinicallytestedNKcellline,NK-92,lacksexpressionofseveralkeyrecep-torsassociatedwithNKcellmaturation,includingCD16.

64

NK-92cellsalsoposesomesafetyconcerns.Asaneoplasticline,NK-92cellsrequireirradiationbeforeadministrationtoavoid

proliferationandestablishmentofleukemiainthehost.64

Inpre-clinicalandclinicalinvestigationsofCAR-Tcelltherapy,extendedinvivolongevityisassociatedwithdurabletumorclearance.SimilarfindingshavebeenreportedinsomeNKcellstudies.

65

,66

IrradiationpreventsNK-92invivoproliferation,whichmayexplainthelimitedeffectivenessofNK-92cellsinvariousclinicaltrials.

67

Duetotheheterogeneityandadaptabilityofcancer,multiplestudieshaveexploredgeneticengineeringstrategiestoenhanceNKcellantitumoractivityandcircumventtumor-mediatedimmunosuppression.Transgenedeliverytechnologyhasimprovedsignificantlyoverthelastdecade,facilitatingtheinves-tigationofeditsatahigh-throughputscaleandthedeliveryofmultipleeditstoasinglecell.

68

GeneeditsinNKcellscangener-allybeclassifiedas‘‘improvingpositivetraits’’or‘‘disruptingnegativetraits.’’Positiveeditsincludetheoverexpressionofchemokinereceptorstopromotelocalizationtotumorsites,theexpressionofcytokinereceptorsforenhancedsurvival,theintroductionofmodifiedCD16αforstrongerADCC,andtheinducedexpressionofdeathreceptors,suchasTRAILR,toheightencytotoxicity.

69

,

70

Oneinnovativeapproachdemon-stratedtheuseoftumor-secretedchemokinestoredirectNKcellsengineeredtoexpressCCR4andCCR2Btothetumorsite.Thisledtotumorreductionandsimultaneousdepletionof

immunosuppressivecellsfromtheTME.

70

NegativeeditsaregainingpopularityduetotheirincreasedeaseofimplementationviaCRISPR-mediatedknockout.TheseincludethedeletionofCISHandGSK3toimproveNKcellmetabolismandthedisruptionofTGFRBIItoalleviatetumor-mediatedimmunosuppression.

20

,65

,71

,

72

RecentadvancementsinCRISPR-mediatedknockin,whichleveragesthecell’sendoge-noushomology-directedrepair(HDR)mechanisms,enablesimultaneouspositiveandnegativeeditingbyinsertinga‘‘posi-tive’’geneofinterestintothelocusofa‘‘negative’’gene.HDRknockinalsoavoidsunwantedgenomicalterations,suchasthosecausedbylentiviraltransfer,bypreciselychoosingtheinsertionsitetoprovidestableandlong-termexpression.

73

,74

Thereareothermethods,outsideofgeneticengineering,toenhancetheantitumoractivityofNKcells.Asmentionedprevi-ously,stimulationofNKcellswithIL-12,IL-15,andIL-18hasbeenusedtogeneratecytokine-inducedmemory-like(CIML)NKcells.

75

,76

ThesecellssecretehighlevelsofIFN-γ,persistlong-term,havepotentantitumoractivity,andhavedemon-stratedexcellentfeasibilityinmanufacturingandreplica-bility.

76

,77

FurtherdevelopmentofimmunotherapystrategiesthatharnessaspectsofNKcellmemorymaybroadlystrengthenNKcelltherapies.

iPSC-DERIVEDNKCELLS

Inrecentyears,iPSCshavegarneredattentionasapromisingapproachtocircumventtheli