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Scien&ficChallengesfortheSafety,EfficacyandQualityofCell-basedTherapeu&cProducts
YojiSATO,PhDHead,DivisionofCell-BasedTherapeu&cProductsNa&onalIns&tuteofHealthSciences,Tokyo,JAPAN
TERMIS-AP2016Session29:MovingRegenera:veMedicinetoBedsideandIndustry(2)
September5,2016
DISCLAIMER:Theviewsandopinionsexpressedinthispresenta;onarethoseofthepresenteranddonot necessarily represent official policy or posi;onof theNa;onal Ins;tute ofHealthSciencesortheMinistryofHealth,Labour&Welfare
NIHSSince1874
NIHS
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PointstoConsideronManufacturingProcess&QualityofCTPs
l Characteriza;onandunderstandingofspecificprofilesofcellsatcri;calsteps(star;ng,intermediate,final)andtheireligibility(differencesinautologous/allogeneic)
l Eligibilityofotherrawmaterialsandmanufacture-relatedsubstancesandtheirqualitycontrol(especially,eligibilityofbiologicalmaterials,noadverseimpactofnon-cellular/;ssuecomponentondesiredcells)
l Verifica;onofmanufacturingprocessandconstancyofmanufacture
l ProductconsistencyintermsofqualityaUributessuchasiden;ty,purity,homogeneityandpotency
l Stability(storagecondi;ons/expira;ondate,freezing&thawingprocesses,andshippingvessel&procedure)
l Qualitycontroloffinalproductthroughrelevantcombina;onofcri;calqualityelementsfromproducts&processaspects
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PointstoConsideronNon-clinicalEfficacy
l Examina;onoffunc;onalexpressions,persistenceofC/Tac;onandtheirexpectedtherapeu;cefficacythroughappropriately-designedtestsusingrelevantanimalsandcells(POC)
l Examina;onoftherapeu;ceffectsusingcell/;ssuemodelsordisease-modelanimals,whereappropriateandpossible
l Indica;onoffarmorepromisingeffectorperformanceoftheproductthanothermedicaltreatments
l Evalua;onofBenefitvs.Risk,consideringaboutPoten;alRiskofProductvs.RealRiskofPa;ent
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PointstoConsideronNon-clinicalSafetyl Presenceofmicroorganism,especiallyViruses
l Tumorigenicity(ESC/iPSC-derivedproducts)
l Inappropriatedifferen;a;on,ectopic;ssueforma;on,undesiredphenotype(SSC/ESC/iPSC-derivedproducts)
l Immunogenicity,immunerejec;onorotherunan;cipatedimmuneresponses(allogeneicCTPs)
l Tes;nginrelevantanimalmodelsorinvitrotoatechnicallypossibleandscien;ficallyreasonableextent,bytakingintoaccountthenatureoftheproductanditstargetdisease.
l Tes;ngcells/;ssuemodelsofanimalorigininrelevantanimalmodels,ifsuchproductmodelsthatcanmimicthoseofhumanareavailable.
l Noadverseimpactofnon-cellular/;ssuecomponentsinstar;ngmaterialsorproducts
l Evalua;onofriskvs.benefit,takingintoconsidera;onaboutpoten;alriskconcernsofproductvs.realriskofpa;ent
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“Tumorigenicity”
Thecapacityofacellpopula;oninoculatedintoananimalmodeltoproduceatumorbyprolifera;onatthesiteofinocula;onand/oratadistantsitebymetastasis.
ReferenceWHOTechnicalReportSeries978Annex3“Recommenda;onsfortheevalua;onofanimalcellculturesassubstratesforthemanufactureofbiologicalmedicinalproductsandforthecharacteriza;onofcellbanks”(2013)
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Interna;onalGuidelinesforTumorigenicityTes;ng
• WHO Technical Report Series 978 Annex 3 “Recommenda&ons for theevalua&on of animal cell cultures as substrates for themanufacture ofbiological medicinal products and for the characteriza&on of cellbanks”(2013)
…isforQCofcellsubstrateslikeCHOcellsandHEK293cells
… and excludes viable animal cells when they are used directly fortherapy by transplanta&on into pa&ents or when they are developedinto cell lines for the purpose of using them as therapeu&c agents bytransplanta&on
• Thereisnointerna&onalguidelinedocumentfortumorigenicitytes&ngofCTPs.
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Newproductmustbeevaluated,basedonnewconcepts
“NewwinemustbeputintonewboUles”
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Purposes of Tumorigenicity(-Associated) Testing for CTPs
1) Quality control of cell substrates (i.g., ESCs, iPSCs) Tumorigenicity is a critical quality attribute of homogeneous cell substrates.
2) Quality control of intermediate/finished products during manufacturing processes The amount of tumorigenic cellular impurities is one of critical quality attributes.
3) Non-clinical safety assessment of finished products Tumorigenicity in the site of administration site is estimated, by in vivo
tumorigenicity testing with immunodeficient animals
・・・LOD is the Key
・・・WHO TRS 978 is applicable
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Sensitivity of Tumorigenicity Testing with Nude Mice (The Method in WHO TRS 978)
after Subcutaneous Administration
The sensitivity is not sufficient for CTPs
TPD50 Fold
Nude 4.0×105 1
NOG 1.3 x 104 25
NOG+Matrigel 7.9 x 10 5,000
Dosetoformatumorin50%oftheanimals
Kusakawa et al., Regen Therapy 2015;1:30-7.
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Nodule Formation16 weeks
-1 0 1 2 3 4 5 6 7 8 9 10
0
20
40
60
80
100
Cells (Log)
Tum
or In
cide
nce
(%) HeLa in Nude
HeLa in NOGHeLa w/ MG in NOG
TPD50 Fold
Nude 4.0×105 1
NOG 1.3 x 104 25
NOG+ Matrigel 7.9 x 10 5,000
In Vivo Tumorigenicity Tests for HeLa Cells with NOG Mice and Matrigel
Kusakawa et al., Regen Therapy 2015;1:30-7.
after Subcutaneous Administration
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Detection of Tumorignic Cellular Impurities (HeLa) in Normal Cells (hMSCs) by NOG mice and Matrigel
Strain GroupTumor incidence at indicated HeLa cell dose at week 16 TPD50
at
week16 0 1×10 1×102 1×103 1×104
NOG HeLa/hMSC(1×106) 0/6 0/6 3/6 6/6 6/6 1.0×102
NOG HeLa/hMSC(1×107) 0/6 1/6 2/6 - (6/6)a 1.8×102
a: Since not all animals inoculated with the highest dose (102) have formed tumors, it was assumed that the tumor
incidence of animals at an even higher dose step (a dummy set of data) would have been 100%.
-: Not tested; ND: Not determined
This method detects HeLa cells in hMSCs at ratios of approx. 1/104 and 1/106, at probabilities of 50% and 17%, respectively.
If the acceptable false negative rate is 1%, sponsors need to confirm no tumor formation in [log0.01/log(1-0.17)=] 25 mice inoculated with 107 hMSCs, to show that the ratio of HeLa-like cellular impurities to hMSCs are less than 1/106.
Kusakawa et al., Regen Therapy 2015;1:30-7.
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Soft Agar Colony Formation Assay
Base Agar Layer
Cell Agar Layer
DMEM/10%FBS
Cell Agar Layer
DMEM/10%FBS
Base Agar Layer
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Detection of Tumorigenic Cellular Impurities (HeLa) in Normal Cells (hMSCs) by Soft Agar Colony Formation Assay
Sog-AgarColonyForma;onAssay(20days) → detected0.1%(1/1000)HeLa/hMSCs※
LLOD2.06inCyQUANTsignalcorrecpondstoLLOD0.02%(1/5000)inHeLa/hMSCs.
Standard curve
0.1 0.2 0.3
-10
0
10
20
30
40
50
LLOD=2.06
HeLa/MSC (%)
Rela
tive
Fold
Cha
nge
0.02%
Y = 151.6*X - 0.4795
*Quan;ta;onbyDNA-BindingDye
(CyQUANT)
Kusakawa et al., Regen Therapy 2015;1:30-7.
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CellPrepara;on SamplePar;;oningColonyCoun;ngbyHigh-ContentImaging
HighlySensi&veMethodforQuan&ta&onofTumorigenicCellularImpuri&esinCTPs
ByDigitalCoun&ngofSingleTumorigenicCells
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
posi;vewell
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
※ASampleImage
“Digital” Soft Agar Colony Formation Assay
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WhenacellsuspensioncontainingasingleHeLacelland107hMSCswasaliquatedinto160wellsandculturedinthesogagarmedia,one“posi;ve”wellwasdetected,
indica;ngitsabilitytodetectaslowas0.00001% (1/10,000,000) HeLacellsinhMSCs.
High-throughputimagingwiththeINCellAnalyzer2000Cellprepara;on:HeLa1/MSC10,000,000 → 160wells(HeLa0.0125/MSC62,500/well)
Kusakawa et al., Sci Rep. 2015; 5: 17892.
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Conclusionsl ThedevelopmentofCTPsisuncertain,becausetheyincludeadvancedand
emergingtechnologieswithlimitedclinicalexperiences.Oneofthebiggestproblemsisthatevalua;ontoolsandapproachestoensuretheirsafety,efficacyandqualityareogenlacking.
l Forexample,tumorigenicityisoneofthemajorconcernsfordevelopingCTPs,par;cularlyhumanES/iPScell-basedproducts.However,nodetailedguidelinehasbeenissuedfortumorigenicitytes;ngforCTPs.
l Weneedtoestablishnewconceptsandtes;ngmethodsfornewQ/E/Sissuesofadvancedproducts.
l Byunderstandingtheabili;esandlimita;onsofeachtes;ngmethod,weshouldselectappropriatemethodsthatmeetthecriteriafordecision-makingduringdevelopmentofCTPs.
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HESI(HealthandEnvironmentalSciencesIns&tute),anon-profit501ccharitableorganiza&on,providestheframeworkforscien&stsfromthepublicandprivatesectorstomeaningfullycollaborateindevelopingscienceforasafer,moresustainableworld.