1、PERSPECTIVEInternational Society for CellularTherapy perspective on immunefunctional assays for mesenchymal stromal cells as potency releasecriterion for advanced phase clinical trialsJACQUES GALIPEAU1,*, MAURO KRAMPERA2,*, JOHN BARRETT3,FRANCESCO DAZZI4, ROBERT J. DEANS5, JOOST DEBRUIJN6, MASSIMO D
2、OMINICI7,WILLEM E. FIBBE8, ADRIAN P. GEE9, JEFFERY M. GIMBLE10, PEIMAN HEMATTI11,MICKEY B.C. KOH12,26, KATARINA LEBLANC13, IVAN MARTIN14,*, IAN K. MCNIECE15,MICHAEL MENDICINO16, STEVE OH17, LUIS ORTIZ18, DONALD G. PHINNEY19,*,VALERIE PLANAT20, YUFANG SHI21,27,*, DAVID F. STRONCEK22,SOWMYA VISWANATHA
3、N23, DANIEL J. WEISS24accepted 17 November 2015)ISSN 1465-3249 Copyright 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.http:/dx.doi.org/10.1016/j.jcyt.2015.11.008Cytotherapy, 2016; 18: 151159AbstractMesenchymal stromal cells (MSCs) as a pharmaceutica
4、l for ailments characterized by pathogenic autoimmune, alloimmuneand inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic spon-sored studies.There is a broad consensus that despite different tissue sourcing and varied culture expansion p
5、rotocols, humanMSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repairfunctionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methodsof measurements of such would be
6、nefit the field.This would satisfy both mechanistic research as well as development of releasepotency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual regis-tration. In response to this unmet need, the International Society for CellularTher
7、apy (ISCT) addressed the issue at an internationalworkshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused ondiscussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune dis-orders.We here
8、provide consensus perspective arising from this forum.We propose that focused analysis of selected MSC markersrobustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Reg-ulatory Authorities.Workshop participants identified three prefer
9、red analytic methods that could inform a matrix assay approach:quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome.We also advocate that potency assays acceptable to the Regulatory Authorities be r
10、endered publiclyaccessible in an “open-access” manner, such as through publication or database collection.KeyWords: Mesenchymal Stromal cells,potency assays,release assays,matrix assays,immune functional testing,clinical trials,ISCTCulture-expanded mesenchymal stromal cells(MSCs) meeting minimal cor
11、e identity for MSCs asdefined by International Society for Cellular Therapy(ISCT) in 2006 1 derived from marrow, adiposetissue, umbilical cord tissue and other sources fromeither autologous or allogeneic donor sources are beingstudied in clinical trials across numerous regulatoryjurisdictions worldw
12、ide.The ailments targeted with thiscell pharmaceutical platform fall roughly within twopathophysiological categories: immune/inflammatoryand tissue repair/restoration 2. It is now widely ac-cepted that the pharmaceutical effect of MSC-like cellsis predominantly mediated by paracrine and contactfacto
13、rs arising from intrinsic MSC physiological pro-cesses that are maintained after culture expansion. Itis further accepted that following in vivo delivery, MSCsare further responsive to environmental cues encoun-tered in situ leading to additional cellular functionalities3. Culture expanded MSC-like
14、cells are unambigu-ously classified as a more-than-minimal-manipulatedcellular and gene therapy (CGT) product regulatedin the United States under section 351 of the PublicHealth Service Act (PHS Act) (42 U.S.C. 262). As atype of CGT product, MSC-like cells require an In-vestigational New Drug Applic
15、ation (IND) from theFood therefore, the adequacy of potencytests is evaluated on a case-by-case basis. The Regu-latory Authorities also recognizes the inherentchallenges in defining potency assays (Table I). Similarguidance has been published by EMA, which definespotency as the quantitative measure
16、of biological ac-tivity based on the attribute of the product, which islinked to the relevant biological properties. Conse-quently, the assay demonstrating the biological activityshould be based on the intended biological effect, whichshould ideally be related to the clinical response andaimed at in
17、vestigating major cellular functions by usingsurrogate markers and appropriate technology.4Thenewly released guidelines of quality control of stemcell products by the Chinese National Health andFamily Planning Commission5mainly emphasizedguidance for facility requirements, biological con-taminants,
18、viability and product consistency. No specificcriterion was given on the issue of potency assay, andthe guidance limits its recommendation to assess-ment of potency according to disease indications.There is no single test that can adequately measureproduct attributes that predict clinical efficacy.T
19、akinginto consideration this limitation, the potency assayshould represent the products mechanism of action(i.e., relevant therapeutic activity or intended biolog-ical effect). However, many CGT products, includingMSC-like cells, have complex (e.g., rely on multiplebiological activities) and/or not
20、fully characterizedmechanisms of action, making it difficult to deter-mine which product attributes are most relevant tomeasuring potency. Indeed, it will be extraordinarilychallenging to perform reductionist mechanistic ex-periments in human subjects that will conclusively definesubstantive MOA of
21、MSC-like cells in vivo and meetmodern standards of ethical conduct in clinical trials.Nonetheless, all attempts should be made to developpotency measurements that reflect the products rel-evant biological properties and that can also serve asa measure of comparability between production lots5.Theref
22、ore, defining hypothesis-driven MOA basedon correlative in vitro experiments and buttressed, wherefeasible, with comparative biology approach in animalsystems will inform the choice of potency assays to bedeveloped.The Regulatory Authorities anticipate thatManufacturers demonstrate clinical effectiv
23、eness bycorrelative “substantial evidence,” that is, evidence thatthe product will have the effect it purports or is rep-resented to have under the conditions of use prescribed,recommended, or suggested in the labeling or pro-posed labeling thereof (section 505(d) of the FDC Act).The traditional app
24、roach for assessing the potency ofbiological products is to develop a quantitative bio-logical assay (bioassay) that measures the activity ofthe product related to its specific ability to affect a givenresult and that also meets the criteria required by Reg-ulatory Authorities (Table II).Analytical
25、methods to measure potencyBioassays can provide a measure of potency by evalu-ating a products active ingredients within a livingbiological system. Bioassays can include in vivo animalstudies, in vitro organ, tissue or cell culture systems,or any combination of these. Development of a quan-titative
26、bioassay for MSC-like products may be4http:/www.ema.europa.eu/docs/en_GB/document_library/Presentation/2015/05/WC500187352.pdf.5http:/ I. Challenges to potency assay development for MSC-likeproducts.Challenges ExamplesInherent variabilityfor startingmaterialsAutologous and allogeneic MSC donorvariab
27、ilityTissue source for MSCs (adipose,marrow, puerperal products)Limited lot size andlimited materialfor testingSingle-dose therapy using autologouscells suspended in a small volumeLimited stability Viability of cell productsFunctionality of cell products at timeof administration relative to banking(
28、thawing)Lack of appropriatereference standardsAutologous cell materialComplex MOA Multiple potential effector functionsof cellsMultiple steps required for functionIn vivo fate ofproductMigration from site of administrationHalf-life of cellular product postadministrationCellular differentiation or ac
29、tivationin to the desired cell typeAdapted from http:/www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/UCM243392.pdf.ISCT perspective on immune functional assays for MSCs 153complicated by properties of the product and/or tech-nica
30、l limitations of certain assays (see Table I). In casesin which development of a suitable bioassay is not fea-sible, it may be necessary to identify a surrogatemeasurement of biological activity. For example, theuse of non-biological analytical assays performedoutside of a living system that is prac
31、tical anddemonstrates adequate performance characteristics forlot release. Examples of such analytical assays pro-vided by the FDA include methods that measureimmunochemical (e.g., quantitative flow cytometry,enzyme-linked immunosorbent assay), molecular (e.g.,reverse transcription polymerase chain
32、reaction, quan-titative polymerase chain reaction, microarray) orbiochemical (e.g., protein binding, enzymatic reac-tions) properties of the product outside of a livingsystem. Analytical assays can provide extensive productcharacterization data by evaluating immunochemi-cal, biochemical and/or molec
33、ular attributes of theproduct. These attributes may be used to demon-strate potency if the surrogate measurement(s) can besubstantiated by correlation to a relevant product-specific biological activity(s).Assay matrixThe FDA states that a single biological or analyticalassay may not provide an adequ
34、ate measure of potency.The following are some potential reasons: (i) producthas complex and/or not fully characterized mecha-nism of action, (ii) product has multiple activeingredients and/or multiple biological activities, (iii)limited product stability, or (iv) biological assay is notquantitative,
35、 not sufficiently robust or lacks precision.If one assay is not sufficient to measure the productattributes that indicates potency, then an alternative ap-proach could be used, such as developing multiplecomplementary assays that measure different productattributes associated with quality, consisten
36、cy and sta-bility. When used together and when results arecorrelated with a relevant biological activity, these com-plementary assays should provide an adequate measureof potency. Such a collection of assays (referred to asan assay matrix) might consist of a combination of bi-ological assays, biolog
37、ical and analytical assays oranalytical assays alone. The assay matrix may includeassays that give a quantitative readout (e.g., units of ac-tivity) and/or qualitative readout (e.g., passfail). Ifqualitative assays are used as part of an assay matrixto determine potency for lot release, stability or
38、 com-parability studies, they should be accompanied by oneor more quantitative assays. A concrete example of anassay matrix for MSC-like cells recognized by the FDAis detailed in a published report by Athersys Inc. (Cleve-land, OH) outlining their effort in establishing anangiogenic potency assay fo
39、r their MultiStem productbased on measure of CXCL5, interleukin (IL)-8 andvascular endothelial growth factor (VEGF) produc-tion coupled to an in vitro cell-based angiogenic assay6. The correlative relationship between the surro-gate measurement and biological activity may beestablished using various
40、 approaches, including com-parison to preclinical/proof of concept data, in vivo data(animal or clinical) or in vitro cellular or biochemicaldata. The suitability of data used to support the cor-relative relationship between the surrogate assay andthe biological activity of a MSC-like product will b
41、eevaluated on a case-by-case basis by the Regulatory Au-thorities and depends on or is influenced by thefollowing: (i) type and relevance of the correlations beingmade, (ii) the amount of product information accu-mulated, (iii) how well the biological activity of theproduct is understood, and (iv) h
42、ow well the surro-gate measurements reflects biological activity.Defining release potency assays for MSC-likecells developed for immunomodulationThe open-ended guidance from the Regulatory Au-thorities and the published precedent of a matrixpotency release assay approach used by Athersys Inc.for Mul
43、tistem in support of their advanced clinicalstudies informs a path forward on how to character-ize a MSC-like product coupling an in vitro bioassayTable II. Release testing of licensed biological product.Release testingApplicable FDA biologics andcGMP regulationsIndicate potency (biologicalactivity/
44、activities) specific tothe product21 CFR 600.3(s) and 610.10;and 21 CFR 210.3(b)(16)(ii)Provide quantitative data 21 CFR 211.194; see also 21CFR 600.3(kk); 21 CFR211.165(d); 211.165(e)Meet pre-defined acceptanceand/or rejection criteria21 CFR 211.165(d); see also 21CFR 600.3(kk); and 21 CFR210.3(b)(
45、20)Include appropriate referencematerials, standards, and/orcontrols21 CFR 210.3(b)(16)(ii) and211.160Establish and document theaccuracy, sensitivity,specificity and reproducibilityof the test methods usedthrough validation21 CFR 211.165(e) and211.194(a)(2)Measure identity and strength(activity) of
46、all activeingredients21 CFR 211.165(a); see also 21CFR 210.3(b)(7)Provide data to establish datingperiods21 CFR 600.3(l) and 610.53(a)Meet labeling requirements 21 CFR 610.61(g)(3) and610.61(r)Adapted from http:/www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Gui
47、dances/CellularandGeneTherapy/UCM243392.pdf.154 J. Galipeau et al.interrogating the cellular secretome by enzyme-linked immunosorbent assay (ELISA) and a functionalcell-based assay. Another industry sponsored exampleis that of Prochymal (Osiris Therapeutics, Inc.), whichis an industrial-scale expand
48、ed MSC-like productderived from marrow collected from random donorsthat was studied as part of prospective randomizedclinical trials. As a surrogate measure of potency, solubleTNFR1 was defined as a release criterion 7.Thereare likely other potency release assays strategies thathave been considered
49、by the Regulatory Authoritiesfor MSC-like ATMPs for use in immunomodulation,but these are unpublished and are not available forpublic consultation because Regulatory Authorities arenot at liberty to publicly disclose otherwise confiden-tial IND disclosure made by Manufacturers (academicor industrial). Nonetheless, these precedents informus that a minimal set of assay components to be takeninto consideration will likely require direct assay(s) ofcell functionalities of MSC-like products and
