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

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

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

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

“Tumorigenicity”　

Thecapacityofacellpopula;oninoculatedintoananimalmodeltoproduceatumorbyprolifera;onatthesiteofinocula;onand/oratadistantsitebymetastasis.

ReferenceWHOTechnicalReportSeries978Annex3“Recommenda;onsfortheevalua;onofanimalcellculturesassubstratesforthemanufactureofbiologicalmedicinalproductsandforthecharacteriza;onofcellbanks”(2013)

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.

Newproductmustbeevaluated,basedonnewconcepts

“NewwinemustbeputintonewboUles”

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

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.

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

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.

Soft Agar Colony Formation Assay

Base Agar Layer

Cell Agar Layer

DMEM/10%FBS

Cell Agar Layer

DMEM/10%FBS

Base Agar Layer

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.

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

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.

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.

HESI（HealthandEnvironmentalSciencesIns&tute）,anon-profit501ccharitableorganiza&on,providestheframeworkforscien&stsfromthepublicandprivatesectorstomeaningfullycollaborateindevelopingscienceforasafer,moresustainableworld.