Identification and selective expansion of functionally superior T cells expressing chimeric antigen receptors

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1 Identifiction nd selective expnsion of functionlly superior T cells expressing chimeric ntigen receptors The Hrvrd community hs mde this rticle openly vilble. Plese shre how this ccess benefits you. Your story mtters. Cittion Published Version Accessed Citble Link Terms of Use Chng, ZeNn L., Pmel A. Silver, nd Yvonne Y. Chen Identifiction nd selective expnsion of functionlly superior T cells expressing chimeric ntigen receptors. Journl of Trnsltionl Medicine 13 (1): 161. doi:1.1186/s doi:1.1186/s July 22, 218 1:2:11 PM EDT This rticle ws downloded from Hrvrd University's DASH repository, nd is mde vilble under the terms nd conditions pplicble to Other Posted Mteril, s set forth t (Article begins on next pge)

2 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 DOI /s RESEARCH Open Access Identifiction nd selective expnsion of functionlly superior T cells expressing chimeric ntigen receptors ZeNn L. Chng 1,2, Pmel A. Silver 3 nd Yvonne Y. Chen 1* Abstrct Bckground: T cells expressing chimeric ntigen receptors (CARs) hve shown exciting promise in cncer therpy, prticulrly in the tretment of B-cell mlignncies. However, optimiztion of CAR-T cell production remins tril-nd-error exercise due to lck of phenotypic benchmrks tht re clerly predictive of nti-tumor functionlity. A close exmintion of the dynmic chnges experienced by CAR-T cells upon stimultion cn improve understnding of CAR T-cell biology nd identify potentil points for optimiztion in the production of highly functionl T cells. Methods: Primry humn T cells expressing second-genertion, nti-cd19 CAR were systemticlly exmined for chnges in phenotypic nd functionl responses to ntigen exposure over time. Multi-color flow cytometry ws performed to quntify dynmic chnges in CAR-T cell vibility, prolifertion, s well s expression of vrious ctivtion nd exhustion mrkers in response to vried ntigen stimultion conditions. Results: Stimulted CAR-T cells consistently bifurcte into two distinct subpopultions, only one of which (CAR hi /CD25 + ) exhibit nti-tumor functions. The use of centrl memory T cells s the strting popultion nd the resilience but not ntigen density of ntigen-presenting cells used to expnd CAR-T cells were identified s criticl prmeters tht ugment the production of functionlly superior T cells. We further demonstrte tht the CAR hi /CD25 + subpopultion upregultes PD-1 but is resistnt to PD-L1-induced dysfunction. Conclusions: CAR-T cells expnded ex vivo for doptive T-cell therpy undergo dynmic phenotypic chnges during the expnsion process nd result in two distinct popultions with drmticlly different functionl cpcities. Significnt nd sustined CD25 nd CAR expression upregultion is predictive of robust nti-tumor functionlity in ntigen-stimulted T cells, despite their correltion with persistent PD-1 upregultion. The functionlly superior subpopultion cn be selectively ugmented by creful clibrtion of ntigen stimultion nd the enrichment of centrl memory T-cell type. Keywords: Chimeric ntigen receptor, CD19 CAR-T cell, T-cell immunotherpy, PD-1 Bckground Adoptive cell therpy using T cells engineered to express tumor-trgeting chimeric ntigen receptors (CARs) is promising tretment strtegy for refrctory diseses such s metsttic melnom, leukemi, nd neuroblstom [1 3]. Severl recent trils hve demonstrted remrkble clinicl efficcy, prticulrly in the tretment of chronic nd cute B-cell mlignncies using CD19- trgeting T cells [4 6]. Accumulting clinicl reports * Correspondence: yvonne.chen@ucl.edu 1 Deprtment of Chemicl nd Biomoleculr Engineering, University of Cliforni Los Angeles, 42 Westwood Plz, Boelter Hll 5531, Los Angeles, CA 995, USA Full list of uthor informtion is vilble t the end of the rticle suggest tht, in ddition to ptient-to-ptient vritions in tumor burden nd overll helth profile, the qulity of individul T-cell products could significntly influence clinicl outcome [5, 7]. However, it remins uncler which T-cell chrcteristics re the most criticl nd predictive of nti-tumor efficcy, nd if nd how such chrcteristics could be promoted during T-cell mnufcturing. As CAR-T cell technology dvnces towrd broder clinicl use, the bility to identify criticl T-cell chrcteristics nd systemticlly optimize CAR-T cell preprtion hs the potentil to significntly improve the robustness of doptive T-cell therpy. 215 Chng et l. This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly credited. The Cretive Commons Public Domin Dediction wiver ( cretivecommons.org/publicdomin/zero/1./) pplies to the dt mde vilble in this rticle, unless otherwise stted.

3 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 2 of 16 Considerble differences exist in the T-cell mnufcturing protocols utilized thus fr by different clinicl groups, rendering efforts to systemticlly optimize the production process prcticl chllenge. Although numerous chrcteriztion ssys nd product relese criteri re pplied to CAR-T cell products, they typiclly generte snpshot views of T-cell properties nd provide limited insight into dynmic chnges experienced by T cells, which my occur throughout in vitro expnsion s well s fter infusion into the ptient. For exmple, phenotypic chrcteristics such s % CD3 +, % CD4 +,%CD8 +,nd%car + re typiclly quntified t the end of cell expnsion prior to product relese for infusion [4 6, 8]. Cytokine production nd cell lysis efficiency re mesured in vitro t single time points to confirm trget-specific functionl ctivity [5, 6, 9]. After doptive trnsfer, in vivo performnce is mesured by quntifying cytokine levels, tumor burden, nd CAR + T-cell count in the ptient [4, 1, 11]. In these chrcteriztion ssys, observed nti-tumor functionlity is ttributed to CAR + T cells s homogenous group, nd time-point dt re used to generlize cross cellexpnsion nd tretment periods. Given tht current clinicl protocols typiclly utilize unsorted, polyclonl T cells for infusion, the ssumption of uniformity mong CAR + T cells is one dictted by experimentl constrints rther thn our understnding of CAR T-cell biology. Indeed, the recognition tht not ll T cells re equl hs prompted ctive reserch on questions such s the optiml T-cell subtype nd cytokine regimen to use for the production of therpeutic T cells [12 16]. However, trilnd-error remins the dominnt pproch to process optimiztion, s typicl chrcteriztion methods such s those described bove provide informtion tht enbles qulity control but not in-depth understnding of how the T cells rrived t their present stte of functionlity or lck thereof. We propose tht close exmintion of dynmic chnges experienced by CAR-T cells throughout stimultion cycle cn provide deeper understnding of CAR T-cell biology nd identify potentil points for optimiztion in the production of highly functionl therpeutic T cells. In this study, we perform quntittive evlutions of the phenotypic nd functionl chnges exhibited by CAR-T cells undergoing ntigen stimultion, including CAR T-cell vibility, prolifertion, s well s the expression of vrious T-cell ctivtion nd exhustion mrkers. Contrry to the ssumption of uniformity, stimulted CAR + T cells consistently bifurcte into two distinct popultions, only one of which (CAR hi /CD25 + ) is functionlly ctive. Detiled in vitro exmintions revel dynmic chnges in CAR-T cells over the course of ntigen stimultion tht re difficult to observe in vivo, nd enble the identifiction of strtegies to mximize the CAR hi /CD25 + cell subpopultion upon ntigen stimultion. Finlly, we demonstrte tht CAR-T cells in the functionlly superior subpopultion upregulte PD-1 but remin functionl when chllenged by trget cells overexpressing PD-L1, indicting n unexpected source of CAR-T cell resilience nd highlighting properties to be considered in the development of combintoril strtegies employing CAR-T cells nd checkpoint therpies. Methods Cell line isoltion nd mintennce Primry humn CD4 + or CD8 + T cells were isolted from helthy donor blood smples obtined from the Boston Children s Hospitl Blood Donor Center nd the UCLA Blood & Pltelet Center. The RosetteSep CD4 + or CD8 + Humn T-cell Enrichment Cocktil (Stemcell Technologies) ws used following mnufcturer s protocols. Centrl memory T cells were obtined by mgnetic bed-bsed sorting. Anti-CD45RA MicroBeds (Miltenyi Biotec) were used to deplete CD45RA + cells, nd CD45RA cells were stined with nti-ccr7-apc (clone G43H7, BioLegend) followed by nti-apc MicroBeds (Miltenyi Biotec) nd enriched for CCR7 + cells. Isolted T cells were seeded t cells/ml in T-cell medi (RPMI-164 medi (Lonz) with 1 % het-inctivted fetl bovine serum (FBS; Life Technologies)) nd stimulted with CD3/CD28 Dynbeds (Life Technologies) t 1:1 cell:bed rtio. Cells were fed 5 IU/ml interleukin (IL)-2 (Chiron) nd 1 ng/ml IL-15 (Miltenyi Biotec) every 48 h nd pssged routinely. H9 nd JeKo-1 cells were obtined from ATCC nd mintined in RPMI-164 with 1 % or 2 % het-inctivted FBS, respectively. K562, TM-LCL, nd Rji cells were generous gifts from Dr. Michel C. Jensen t Settle Children s Reserch Institute nd mintined in T-cell medi. CD19 + K562 nd CD19 + H9 cells were generted by lentivirlly trnsducing prentl K562 nd H9 cells with CD19 cdna expressed from n EF1α promoter nd sorting for CD19 + cells by fluorescence-ctivted cell sorting (FACS). Genertion nd isoltion of CAR-expressing T cells Lentivirus ws produced s previously described [17]. T-cells stimulted with CD3/CD28 Dynbeds for 3 dys were trnsduced t multiplicity of infection of 3 with T cells/5 μl/well in 24-well plte nd supplemented with 5 IU/ml IL-2, 1 ng/ml IL-15, nd 5 μg/ml polybrene (Sigm Aldrich). No virus ws dded to the mock-trnsduced control. The plte ws centrifuged t 8 g for 3 min t room temperture with slow ccelertion nd no brke. Cells were fed fresh medi with cytokines on dy 2 post trnsduction, wshed on dy 3, nd mintined s described bove until Dynbed removl on dy 6 post trnsduction. To obtin EGFRt + (CAR + ) popultions, trnsduced cells were stined with biotinylted Erbitux (Bristol-Myers Squibb; biotinylted in house) followed by mgnetic sorting using nti-biotin

4 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 3 of 16 MicroBeds (Miltenyi Biotec) ccording to the mnufcturer s protocols. CAR + T-cell frctions with different CAR expression levels were isolted by stining trnsduced cells with biotinylted Erbitux followed by streptvidin-pe (Jckson Immunoreserch), then sorted by FACS. Regrdless of sorting method, CAR + cells were expnded s previously described [18]. Briefly, T cells were resuspended in 5 ml totl volume with γ-irrdited (8 Gy) TM-LCL cells nd supplemented with 5 IU/ml IL-2 nd 1 ng/ml IL-15 every 48 h. Stimulted high nd low CAR-expressing popultions were isolted by FACS fter 2 h of co-incubtion with CD19 + K562 trget cells t 2:1 effector-to-trget (E:T) rtio. Surfce mrker stining For surfce mrker stining, T cells were seeded in 96-well pltes with indicted trget cells (unirrdited) t 2:1 E:T rtio unless otherwise noted. Experiments with T cells were performed in 24-well pltes. When indicted, γ-irrdited (1 Gy) K562 trgets were used. When indicted, CD28 monoclonl ntibody (clone CD28.2; ebiosciences) ws pplied t 1 μg/ml to provide CD28 costimultory signl. Cell mixtures were incubted t 37 C, nd nlyzed t the indicted time points with fluorescently lbeled monoclonl ntibodies binding CCR7 (clone REA18), CD19 (clone LT19), CD25 (clone BC96), CD27 (clone M-T271), CD45RA (clone T6D11), CD57 (clone TB3), PD-1 (PD ), PD-L1 (clone 29E.2A3), nd Tim-3 (clone F38-2E2) (BioLegend nd Miltenyi Biotec). V5-conjugted Annexin V (BD Biosciences) nd Pcific Blue-conjugted Annexin V (BioLegend) were used to detect pre-poptotic cells. CAR expression ws probed with Protein L (Genscript) followed by PE-conjugted streptvidin(jcksonimmunoreserch)orwithapc-conjugted polyclonl ntibody binding humn IgG Fcγ (Jckson Immunoreserch). EGFRt expression ws probed with biotinylted Erbitux followed by PE-conjugted streptvidin. Anlyses were performed on MACSQunt VYB flow cytometer (Miltenyi Biotec) equipped with 45-, 488-, nd 561-nm lsers. Dt were processed using FlowJo softwre (TreeStr). Cell prolifertion nd cytokine production quntifiction T cells were lbeled with.2 μm crboxyfluorescein dicette succinimidyl ester (CFDA-SE, Life Technologies) for cell prolifertion trcking. A humn Th1/Th2 cytokine cytometric bed rry kit (BD Biosciences) ws used ccording to the mnufcturer s protocols to quntify cytokine secretion. Co-incubtions with trget cells were set up s described bove. Smples were nlyzed with MACSQunt VYB flow cytometer nd cytokine production ws quntified using the FCAP Arry 3. softwre (Soft Flow). Quntittive PCR Genomic DNA nd cdna were isolted from frozen T-cell pellets with DNesy Blood nd Tissue Kit (Qigen) nd SuperScript III CellsDirect cdna Synthesis System (Life Technologies), respectively. Quntittive PCR (qpcr) ws performed using SsoFst EvGreen Supermix (Bio Rd), CFX96 Rel-Time Therml Cycler (Bio Rd), nd WPRE forwrd nd reverse primers (5 -TTTCCGGGA CTTTCGCTTTC nd 5 -AAGGGACGTAGCAGAAG GAC, respectively, for cdna; or 5 -ACTGTGTTTGCT GACGCAACCC nd 5 -CAACACCACGGAATTGTCA GTGCC, respectively, for gdna) ccording to the mnufcturer s protocols. The reference β-ctin primer set 5 -TCCCTGGAGAAGAGCTACGA (forwrd) nd 5 -AG CACTGTGTTGGCGTACAG (reverse) provided normliztion control. The qpcr protocol included 3 cycles of 5-s denturtion step t 95 C for cdna nd 98 C for gdna, nd 5-s nneling/extension step t 6 C. All rections were performed in qudruplictes, nd threshold cycle (Ct) vlues were verged to obtin the rithmetic men. Reltive WPRE levels were clculted with the following formul: ð RL¼ ε ActinÞ C t; Actin ð Þ C t; WPRE ε WPRE where RL indictes reltive WPRE levels, ε x indictes primer efficiency for gene x, nd C t,x indictes the verged Ct vlue for gene x. Stndrd devition in reltive WPRE levels ws clculted with the following formul: qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi s:d:¼ ðrl lnε Actin Þ 2 ðs:d: Actin Þ 2 þðrl lnε WPRE Þ 2 ðs:d: WPRE Þ 2 where s.d. x indictes the stndrd devition clculted from the qudruplicte smples for gene x. Sttisticl methods Dt re presented s mens ± stndrd devitions s stted in figure legends. Results were nlyzed by twotiled unpired Student s t test with simple Bonferroni correction for multiple comprisons when pproprite. Tests were conducted with sttisticl significnce set t p <.5. Results Antigen stimultion results in the emergence of CAR hi cells To investigte the degree of heterogeneity mong CAR-T cells, CAR constructs were stbly integrted into bulk primry humn CD8 + T cells vi lentivirl trnsduction. Unless otherwise indicted, n nti-cd19 CAR contining CD28 s the co-stimultory domin ws used [19], nd truncted epiderml growth fctor receptor (EGFRt) ws linked to the CAR vi T2A peptide (Fig. 1). In this configurtion, CAR nd EGFRt re trnscribed s single mrna but trnslted into two seprte proteins, llowing

5 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 4 of 16 T2A 5 LTR RRE CD19 scfv IgG4 Hinge CD28 tm/cyto CD3 ζ EGFRt WPRE 3 LTR P EF1α CAR hi but not CAR lo cells exhibit robust T-cell functions Given their differences in CD25 expression, we hypothesized tht the CAR hi subpopultion is productively ctivted while CAR lo cells remin inctive or re nergized. During co-incubtion with CD19 + trget cells in the bsence of exogenous cytokines, CD8 + T cells expressing the CD19 CAR undergo rpid nd drmtic initil decline in vible T-cell count, followed by popultion rebound in which CAR hi cells emerge s the dominnt T-cell group (Fig. 2), consistent with the hypothesis tht only CAR hi cells re productively ctivted. In contrst, mock-trnsduced T cells s well s CAR-T cells coincubted with prentl (CD19 ) K562 cells show grdul decline in vible T-cell count without chnges in CAR expression level, indicting tht the dynmic popultion chnges observed in Fig. 2 re specificlly triggered by ntigen stimultion. Primry CD4 + CAR-T cells s well s mixed CD4 + nd CD8 + CAR-T cells show the sme ptterns of popultion dynmics (Additionl file 3: Figure S3), demonstrting tht the behvior is not unique to the CD8 + phenotype nd is lso representtive of mixed CD4 + nd CD8 + T cells typiclly used in therpeutic settings. CFSE dilution nd Annexin V stining results demonstrte tht CAR hi cells proliferte robustly with miniml poptosis; in contrst, CAR lo cells divide t the sme low rte s unstimulted CAR-T cells, nd exhibit incresed cell deth compred to both CAR hi nd unstimulted cells (Fig. 2b, c). These results further support the hypothesis tht the CAR lo cells, despite expressing secondb c d Anti-CD19 (CD28) CAR Anti-CD19 (4-1BB) CAR Anti-CD2 (4-1BB) CAR Anti-CD19 (CD28) CAR e EGFRt CD25 CD19 + K562 Prentl K562 CD19 + K562 Prentl K562 CD2 + K562 Prentl K562 Prentl K562 + Dynbeds Prentl K562 Fig. 1 ACAR hi /CD25 + popultion emerges upon ntigen stimultion of CAR-T cells. Schemtic of the second-genertion nti-cd19 CAR with CD28 co-stimultory domin; cyto: cytoplsmic domin; EGFRt: truncted EGFR peptide; LTR: long terminl repets; RRE: Rev responsive element; tm: trnsmembrne domin; scfv: single chin vrible frgment; T2A: Those sign virus 2A peptide; WPRE: woodchuck heptitis virus posttrnscriptionl response element. b-e CD8 + T cells expressing vrious CARs seprte into CAR hi /CD25 + nd CAR lo /CD25 popultions within 24 h of ntigen or CD3/CD28 stimultion. T cells expressing nti-cd19 CARs with b CD28 or c 4-1BB co-stimultion domin or d n nti-cd2 CAR with 4-1BB ll result in popultion polriztion upon stimultion with K562 trget cells expressing cognte ntigens. e CAR T-cell stimultion with mgnetic beds coted with nti-cd3 nd nti-cd28 ntibodies lso yields bifurcted popultions quntifiction of CAR expression vi ntibody stining of surfce-bound EGFRt, without disrupting potentil interctions between CAR molecules nd their lignds [2]. Costining experiments confirmed tht EGFRt stining tightly correltes with direct CAR stining in both stimulted nd unstimulted CAR-T cells (Additionl file 1: Figure S1). EGFRt is prticulrly useful to trck the expression of the nti-cd19 CAR in this study becuse the nti-cd19 scfv used is only wekly stined by regents such s protein L nd nti-fb ntibody tht typiclly bind scfvs. Unless specified otherwise, we hve used EGFRt to mesure CAR expression in the studies reported here. CAR-T cells were co-incubted with prentl (CD19 ) or CD19 + K562 trget cells nd monitored for five dys. A CAR hi /CD25 + group with elevted CAR expression consistently emerged within 24 h of co-incubtion, resulting in popultion distinct from the CAR lo /CD25 group, which mintined the originl CAR expression level (Fig. 1b d). This popultion bifurction is observed in both CD4 + nd CD8 + T cells (Fig. 1b nd Additionl file 2: Figure S2), is independent of the co-stimultory signls present in the CAR (either CD28 or 4-1BB; Fig. 1b, c), nd is not restricted to prticulr ntigen (either CD19 or CD2; Fig. 1c, d). We lso observed the emergence of CAR hi cells fter stimulting CAR-T cells with CD3/CD28 beds (Fig. 1e), indicting tht the popultion bifurction is not unique to ntigen presenttion by K562 cells or to the CAR signling domin. Insted, it lso occurs when T cells re stimulted vi the endogenous T-cell receptor (TCR)/CD28 mchinery. In ll instnces studied, CAR lo cells exhibited low or no expression of the ctivtion mrker CD25 while CAR hi cells showed robust CD25 upregultion (Fig. 1b e), suggesting distinct ctivtion sttes for the CAR hi nd CAR lo popultions.

6 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 5 of 16 # Vible T Cells b Medin CFSE (FU) CD8 CAR with CD19 + K562 CD8 CAR with Prentl K562 CD8 Mock with CD19 + K562 45, 1 45, 1 45, 3, 15, % of Totl # Vible T Cells 3, 15, # Vible T cells CAR hi, CD19 + K562 CAR lo, CD19 + K562 CAR-T cells, Prentl K % CAR hi c % Annexin V % of Totl % CAR lo # Vible T Cells 3, 15, CAR hi, CD19 + K562 CAR lo, CD19 + K562 CAR-T cells, Prentl K562 Fig. 2 Antigen-stimulted CAR-T cells undergo selective expnsion of the CAR hi comprtment. CFSE-lbeled, CD8 + CAR-expressing cells or mock-trnsduced (CAR ) T cells were co-incubted with prentl (CD19 )orcd19 + K562 trgets without exogenous cytokines nd monitored for totl T-cell count (left red xis) nd CAR hi or CAR lo s frction of totl CAR-T cells (right blck xis), b medin CFSE intensity, nd c Annexin V stining. Averge vlues of triplictes re shown with error brs indicting ± 1 stndrd devition (s.d.) genertion CAR contining co-stimultion domin, hve not been properly ctivted. In ddition to differences in prolifertive potentils, CAR hi cells exhibit clerly superior functionl profiles compred to CAR lo cells. The two CD19 CAR-T cell popultions were seprted by FACS fter 2 h of co-incubtion with CD19 + K562 trget cells. Multiplex cytokine mesurements reveled robust Th1 cytokine production by CAR hi cells during the 24-h period following cell sorting (Fig. 3). In contrst, CAR lo cells produced reltively high levels of IFN-γ but not TNF-α or IL-2, consistent with previous study reporting the bility of nergic T cells to produce IFN-γ but not IL-2 [21] (Fig. 3). CAR-T cells hve been reported to serve s seril killers of tumor cells in successfully treted ptients [4], nd the bility to mintin functionlity in the fce of high tumor burden nd repeted stimultion is criticl to the therpeutic efficcy of CAR-T cells. Upon reexposure to trget cells, sorted CD8 + CAR lo T cells showed miniml trget-cell lysis (Fig. 3b). In contrst, CD8 + CAR hi T cells rpidly eliminted CD19 + K562 trgets, chieving even more complete trget clernce thn unsorted CAR-T cells (Fig. 3b). Both T-cell popultions underwent contrction in vible T-cell count upon ntigen exposure, but CAR hi cells gretly outperformed CAR lo cells in subsequent prolifertion (Fig. 3c). pg/ml c # Vible T Cells 6, 4, 2, 75, 5, 25, IFN-γ TNF-α IL-2 IL-1 IL-6 IL CAR hi CAR lo CAR hi CAR lo b d # Vible Trget Cells pg/ml 4, 3, 2, 1, 6, 4, 2, CAR hi /Prentl K562 CAR hi /CD19 + K562 CAR hi CAR lo IFN-γ TNF-α IL-2 IL-1 IL-6 IL-4 CAR lo /Prentl K562 CAR lo /CD19 + K562 Unsorted CAR Fig. 3 CAR hi nd CAR lo T cells exhibit distinct functionl cpbilities. CAR hi nd CAR lo cells were sorted nd subsequently cultured without ntigen stimultion or exogenous cytokines. Cytokine production ws mesured 24 h post sorting. b-d Sorted CAR hi nd CAR lo cells were co-incubted with CD19 + K562 trgets without exogenous cytokines nd monitored for b vible trget-cell count, c vible T-cell count, nd d cytokine production fter 24 h of co-incubtion. Sorted CAR lo vlues in b nd c re from single smples due to the rrity of vible cells recovered for this popultion from cell sorting. For ll other smples, verge vlues of triplictes re shown with error brs indicting ± 1 s.d.

7 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 6 of 16 Furthermore, CAR hi but not CAR lo cells robustly secreted the Th1 cytokines TNF-α nd IL-2 upon ntigen re-chllenge (Fig. 3d). As previously observed, CAR lo cells retined the bility to produce IFN-γ (Fig. 3d). The contrsts between CAR hi nd CAR lo cells described bove were lso observed mong CD4 + CAR-T cells (Additionl file 4: Figure S4), indicting the functionl disprities observed re intrinsic to CAR hi vs. CAR lo subpopultions nd re not restricted to the CD8 + phenotype. The cler functionl superiority of CAR hi cells suggests tht mximiztion of CAR hi cells within given CAR-T cell preprtion my enhnce the ntitumor potentil of the cell product. We next explored the source of the CAR hi phenotype nd methods to direct the T cell popultion towrd this functionl subset. The CAR hi phenotype is trnsient stte of ctivtion induced by ntigen stimultion Efforts to mximize CAR hi cells require knowledge of their origin. One possibility is tht CAR hi cells re geneticlly encoded with higher CAR copy numbers, nd their superior prolifertive cpbility enbles their rpid enrichment fter ntigen stimultion, despite their pprent bsence from the originl CAR + T-cell popultion. Alterntively, CAR hi cells my be geneticlly similr to CAR lo cells, but productive stimultion results in distinct, ctivted stte. To distinguish between these two possibilities, CD8 + T cells were sorted into three popultions with different CAR expression levels prior to ntigen stimultion (Additionl file 5: Figure S5). After cell expnsion over 9 dys in culture, qpcr performed on genomic DNA confirmed significnt differences in the copy number of the CAR trnsgene in the three popultions (Fig. 4). Upon ntigen stimultion, ll three popultions were ble to generte CAR hi cells with clerly elevted CAR expression (Fig. 4b). In fct, T cells with the lowest CAR genomic copy number yielded the highest % CAR hi nd the highest CD25 expression level mong CAR hi cells upon ntigen stimultion (Fig. 4b), indicting tht high genetic copy number of the CAR trnsgene is neither essentil nor utomticlly conducive to the emergence of the CAR hi phenotype. Quntittive PCR performed on sorted CAR hi nd CAR lo cells showed tht CAR hi nd CAR lo cells differ slightly in genomic copy numbers of the CAR construct (Fig.5).However,thetwopopultionsdivergemore prominently in CAR trnscription levels, with CAR hi cells overexpressing the nti-cd19 CAR mrna by 3.5 folds (CD4 + T cells) or 7.5 folds (CD8+ T cells) compred to CAR lo cells(fig.5).inddition,carexpression levels in CAR hi cells return to bseline (i.e., the sme level s unstimulted nd CAR lo cells) within dys fter the removl of ntigen stimultion (Fig. 5b), indicting tht the CAR hi phenotype is predominntly due to trnsient upregultion of CAR expression. Furthermore, ntigen-stimulted CAR-T cells upregulte the surfce expression of both CAR nd EGFRt, which re co-trnscribed s one mrna but trnslted into two Reltive Genomic Copy # x Lowest Gte 2.81 x Middle Gte 4.72 x Highest Gte b Low Copy # Mid Copy # High Copy # CD19 + K562 No trget EGFRt CD25 Fig. 4 CAR hi phenotype does not require high genomic copy number of CAR trnsgene. Reltive genomic CAR copy number in three sorted CAR T-cell popultions s determined by quntittive PCR reltive to the housekeeping gene β-ctin. The lowest copy number is set to 1. Vlues re verges of qudruplictes with error brs indicting ± 1 s.d. b CAR T-cell popultions with vrying genomic CAR copy number seprte into CAR hi /CD25 + nd CAR lo /CD25 popultions within 18 h of ntigen simultion

8 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 7 of 16 c Reltive Copy Number Fold Chnge: b Medin EGFRt (FU) Meidn EGFRt (FU) CD4 gdna CD8 Reltive Expression CD Dys Post-Sort CD19 + K562 cdna CD No Trget Cells CD8 CAR hi CD4 CAR hi CD4 CAR lo CAR lo CAR hi seprte proteins (Fig. 5c nd Additionl file 1: Figure S1D). This result indictes tht the increse in CAR surfce expression is not due to CAR-specific chnges in post-trnsltionl processes such s surfce locliztion, receptor endocytosis, or protein degrdtion. Tken together, these dt indicte tht elevted CAR expression in CAR hi cells is minly result of trnsient CAR trnscript upregultion in response to cell ctivtion, nd tht incresing the copy number of the CAR trnsgene is unlikely to be the most effective mens of promoting the CAR hi phenotype. Medin scfv (CAR) (FU) Fig. 5 CAR hi T cells rise from trnsient CAR trnscript upregultion. Genomic copy number nd mrna expression of CAR hi nd CAR lo popultions s determined by quntittive PCR reltive to the housekeeping gene β-ctin. The lowest expression level or copy number is set to 1. b EGFRt expression level of sorted cells mintined in culture with exogenous IL-2 nd IL-15 nd no ntigen stimultion. c EGFRt nd CAR surfce expression on CD8 + CAR-T cells s mesured by Erbitux nd Protein-L stining, respectively, over 4 dys of co-incubtion with or without CD19 + K562 trget cells. Vlues in b re verges of qudruplictes with error brs indicting ± 1 s.d.; ll other plots show verge vlues of triplictes with error brs indicting ± 1 s.d. Overstimultion results in loss of the CAR hi phenotype One pproch to mximizing CAR hi cells is to prevent the dysfunctionl CAR lo stte. Given tht the CAR hi phenotype is induced by ntigen stimultion, we next investigted whether the CAR lo cells fil to upregulte CAR expression becuse they experienced indequte stimultion or if they becme exhusted due to overstimultion. When sorted CAR hi cells were re-chllenged with ntigen, the mjority retined elevted CAR expression but smll popultion fell into the CAR lo gte (Fig. 6), suggesting tht repeted ntigen exposure nergized minority of the CAR hi cells. Menwhile, lrge portion of CAR lo cells experienced further reduction in CAR expression nd no cells moved into the CAR hi regime upon ntigen rechllenge (Fig. 6). These results support the hypothesis tht sufficiently strong stimultion is required for the CAR hi phenotype, but overstimultion results in the exhustion of ctivted cells nd decline into the dysfunctionl CAR lo /CD25 stte. Additionl ntigen chllenge to CAR lo cells would only further decrese CAR expression nd T-cell functionlity. These results re consistent with our previous observtion tht T cells with the highest genomic copy number of the CAR trnsgene (nd thus the highest cpcity to receive ntigen stimultion) yielded the lrgest proportion of CAR lo cells fter ntigen stimultion (Fig. 4b). Therefore, mximiztion of the CAR hi popultion requires precise clibrtion of ntigen stimultion, nd systemtic pproch to this tsk would fcilitte the optimiztion of the cell production process. Persistent ntigen stimultion fcilittes CAR hi expnsion Severl stimultion conditions were evluted to determine n effective protocol for the preprtion of T cells with the CAR hi phenotype. Since co-stimultion plys mjor role in chieving productive T-cell ctivtion, we first exmined whether dditionl co-stimultion could enhnce CAR hi development nd forestll the emergence of non-functionl CAR lo cells. However, supplementing CD28 gonist ntibody to CAR-T cells immeditely prior to ntigen exposure did not significntly lter the CAR hi vs. CAR lo distribution dynmics nor impct the bsolute CAR hi cell numbers (Additionl file 6: Figure S6). T-cell expnsion with the id of ntigen-presenting feeder cells is well-estblished method for CAR-T cell preprtion [18, 22, 23]. We next evluted multiple feeder cell lines for their bility to support CAR hi /CD25 + cell genertion (Fig. 7 c). CD8 + CAR-T cells were coincubted t 2:1 effector-to-trget (E:T) rtio with severl CD19 + trget cell lines, including nturlly CD19 + JeKo-1, Rji, nd TM-LCL cells, s well s K562 nd H9 cells modified to stbly express CD19. Besides JeKo-1 cells, which were depleted rpidly, ll trget cell lines tested resulted in the emergence of CAR hi cells, nd CAR hi expression ws lwys correlted with CD25

9 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 8 of 16 Prentl K562 CD19 + K562 CAR lo CAR hi Normlized to Mode Normlized to Mode EGFRt Normlized to Mode Normlized to Mode EGFRt EGFRt EGFRt Fig. 6 Antigen re-chllenge results in CAR expression reduction. Sorted CD8 + CAR hi nd CAR lo cells were ssyed for EGFRt surfce expression fter 24 h of co-incubtion. A smll frction of CAR hi cells becomes CAR lo upon ntigen re-chllenge, while significnt reduction in CAR expression is observed mong CAR lo cells fter stimultion # Vible Trget Cells 5, 4, 3, 2, 1, b # CAR hi Cells 1, 7,5 5, 2, c Medin CD25 (FU) of CAR hi Cells CD19 + K562 TM-LCL CD19 + H9 Rji JeKo-1 d # CAR hi Cells 6, 5, 4, 3, 2, 1, e Medin CD25 (FU) Among CAR hi Cells :2 2:1 5:1 1: Fig. 7 Vrying CAR-T cell stimultion conditions impcts the dynmics of the CAR hi response. -c CD8 + CAR-T cells were co-incubted with vrious trget cell lines t 2:1 E:T rtio nd monitored for trget cell counts, b number of CAR hi T cells, nd c CD25 expression mong CAR hi cells. d-e CD8 + CAR-T cells were co-incubted with CD19 + K562 t vrious E:T rtios nd monitored for d number of CAR hi cells nd e CD25 expression mong CAR hi cells. Averge vlues of triplictes re shown with error brs indicting ± 1 s.d.

10 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 9 of 16 upregultion (Additionl file 7: Figure S7). However, not ll CAR hi cells upregulted the ctivtion mrker eqully (Fig. 7c). Considering both the number of CAR hi /CD25 + cells nd the intensity of CD25 expression (Fig. 7b, c), the results suggest CD19 + K562 nd TM-LCL cells re both suitble cndidtes s feeder cells for CD19 CAR-T cell expnsion. Interestingly, these two cell lines re lso the most resistnt to CAR-T cell medited lysis mong the trget cell lines tested (Fig. 7). In contrst, ntigen expression level on trget cells shows no correltion with the trget cells susceptibility to T-cell medited lysis or with CAR hi cell development (Additionl file 8: Figure S8). Therefore, it is the persistence of ntigen presenttion rther thn the ntigen density on individul trget cells tht predicts CAR hi emergence ptterns. The most resilient trget cell line, CD19 + K562, resulted in reltively smll CAR hi popultion t erly time points, but the totl number nd CD25 expression level of CAR hi cells incresed stedily throughout subsequent dys, confirming CD19 + K562 s n effective trigger for the functionl CAR hi phenotype (Fig. 7 c). To more precisely evlute the impct of trget-cell dosge on CAR T-cell bifurction, we co-incubted CD8 + CAR-T cells with CD19 + K562 trgets t 1:2, 2:1, 5:1, nd 1:1 E:T rtios. Results corroborte the observtion tht sustined production of CAR hi cells requires high trgetcell inputs tht enble persistent ntigen presenttion (Fig. 7d). CAR-T cells treted with the lrgest number of ntigen-presenting cells ultimtely resulted in the highest medin CD25 expression level mong CAR hi cells, but only fter stedy rise over time s previously observed (Fig. 7e). These observtions hold true regrdless of whether the trget cells were irrdited prior to coincubtion with CAR-T cells (Additionl file 9: Figure S9), confirming the pplicbility of this evlution method to CAR-T cell expnsion protocols employed in clinicl settings. CAR hi cells re PD-1 + but resist PD-L1-induced dysfunction The observtion tht sustined ntigen stimultion is required for the mintennce of the CAR hi phenotype (Figs. 5b nd 7b, d) rises the question of whether CAR hi cells re t risk of exhustion, leding to lower therpeutic efficcy despite their functionl cpbilities in vitro. Indeed, surfce ntibody stining reveled tht CAR hi cells upregulte PD-1, mrker whose sustined expression is generlly ssocited with T-cell dysfunction [24] (Fig. 8). Furthermore, CAR hi cells generted through stimultion by the most resilient ntigen-presenting trget cells (CD19 + K562) nd the highest ntigen concentrtion (1:2 E:T rtio) lso express the highest levels of PD-1 (Fig. 8). Although it is unsurprising tht ntigenstimulted cells upregulte PD-1, our trget-cell lysis, T-cell prolifertion, nd cytokine production ssy results b% PD-1+ Among CARhi Cells % PD-1 + Among CAR hi Cells CD19 + K562 TM-LCL CD19 + H9 Rji 1:2 2:1 5:1 1:1 Fig. 8 PD-1 is upregulted in CAR hi cells with intensities dependent upon stimultion conditions. CD8 + CAR-T cells were co-incubted with vrious trget cell lines or b CD19 + K562 trget cells t vrious E:T rtios. % PD-1 + mong CAR hi cells ws determined by surfce ntibody stining. Averge vlues of triplictes re shown with error brs indicting ± 1 s.d. did not revel ny sign of dysfunction mong the PD-1 + CAR hi cells (Fig. 3), contrry to previous reports on PD-1 + tumor-trgeting T cells [25 28]. One possible explntion is tht the trget cell line used in this study (K562) did not express high levels of PD-L1 (Additionl file 1: Figure S1A). To investigte this possibility, the CD19 + K562 trget line ws engineered to stbly overexpress PD-L1 (Additionl file 1: Figure S1) nd used in weeklong co-incubtion ssys with both CD4 + nd CD8 + CD19 CAR-T cells. CD8 + CAR-T cells monitored over seven dys of coincubtion in the bsence of exogenous cytokines showed no susceptibility to high PD-L1 expression on trget cells, demonstrting little to no chnges in % CAR hi, % Annexin V +, CD25 nd Tim-3 expression, T-cell prolifertion, nd trget-cell lysis efficiency (Fig. 9). A smll but sttisticlly significnt reduction in PD-1 expression ws observed in CD8 + CAR-T cells upon co-incubtion with PD-L1 + trget cells (Fig. 9e). CAR hi expression ws consistently correlted with incresed CD25, PD-1, nd Tim-3 expression (Additionl file 11: Figure S11), but high levels of PD-L1 on trget cells did not result in reduced prolifertive or trget-cell lysis response by CD8 + CAR-T cells over the 7-dy observtion period (Fig. 9f, g). It should be noted tht since CD8 + CAR-T cells re cpble of eliminting

11 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 1 of 16 b c d % CAR hi % Annexin V % CD25 + % Tim CD8 + CAR-T Cells CD8 + CAR-T Cells CD8 + CAR-T Cells CD8 + CAR-T Cells % CAR hi % Annexin V + % CD25 + % Tim CD4 + CAR-T Cells CD4 + CAR-T Cells CD4 8 + CAR-T Cells CD4 + CAR-T Cells e f g Trget Cell # % PD-1 + CD8 + CAR-T Cells 5 4 * , 5, 4, 3, 2, 1, CD8 + CAR-T Cells CD8 + CAR-T Cells CD19 + PD-L1 + K562 CD19 + K562 Trget Cell # % PD CD4 + CAR-T Cells , 4, 3, 2, 1, CD4 + CAR-T Cells CD4 + CAR-T Cells Fig. 9 CAR-T cells re minimlly impcted by PD-L1 expression on trget cells. CD8 + nd CD4 + CAR-T cells were co-incubted with CD19 + or CD19 + / PDL1 + K562 trget cells without exogenous cytokines nd monitored for %CAR hi, b %AnnexinV +, c %CD25 +, d %Tim-3 +, e %PD-1 +, f CFSE intensity, nd g trget cell count. An sterisk indictes significnt differences compring co-incubtions with CD19 + versus CD19 + /PDL1 + K562 trget cells t tht time-point s determined by two-tiled unpired Student s t tests with the Bonferroni correction (p =.19 in e nd p =.76 in g). Blck rrows denote the ddition of trget cells to ensure sustined exposure to PD-L1. Averge vlues of triplictes re shown with error brs indicting ± 1 s.d. Medin CFSE (FU) Medin CFSE (FU) * CD19 + K562 trget cells, dditionl trget cells were dded to the co-incubtion culture t 48 nd 96 h to ensure sustined exposure of CAR-T cells to PD-L1 presenttion by trget cells. This continuous PD-L1 presenttion did not result in noticeble impct on CD8 + CAR-T cell function. However, the repeted ntigen chllenge did result in decline in CAR hi cells reltive to CAR lo cells t 144 h (Fig. 9), consistent with our previous observtion tht overstimultion contributes to the loss of the functionl CAR hi popultion (Fig. 6). PD-L1 expression protected trget cells from CD4 + CAR T-cell lysis t erly time points (Fig. 9g), potentilly due to the higher PD-1 expression level in CD4 + cells compred to CD8 + cells (Additionl file 12: Figure S12). However, the resistnce to CD4 + T-cell medited lysis exhibited by PD-L1 + trget cells ppered to be temporry, nd PD-L1 expression on trget cells did not impct ny of the other T-cell prmeters quntified (Fig. 9). No trget cells beyond the initil input were dded to the CD4 + culture since CD4+ CAR-T cells did not eliminte the originl trget popultion. Tken together, the results indicte tht CAR hi cells persistently upregulte PD-1 expression but re not functionlly impired by PD-L1 presenttion on K562 trget cells. CAR-T cells generted from T CM subset re primed for the CAR hi /CD25 + phenotype As demonstrted bove, ntigen stimultion is n integrl prt of CAR-T cell preprtion nd its clibrtion cn significntly influence the efficiency of CAR hi genertion. Another importnt prmeter in CAR-T cell preprtion is the specific subtype of T cells used to mke CAR-T cells. Surfce ntibody stining reveled tht the CAR hi popultion is enriched in centrl memory T (T CM ) cells while the CAR lo popultion consists of predominntly effector (T E ) nd effector memory T (T EM ) cells (Fig. 1). We thus investigted whether enrichment for T CM cells prior to CAR trnsgene trnsduction my increse the CAR-T cells potentil to ttin the CAR hi phenotype. From the sme helthy donor s blood smple, bulk CD8 + T cells nd CD8 + T CM (CCR7 + /CD45RA ) cells were isolted seprtely (Additionl file 13: Figure S13A),

12 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 11 of 16 1 CD8 + T cells 1 CD4 + T cells % T-cell Subtype % T-cell Subtype CAR: K562: +(hi) +(lo) CAR: K562: +(hi) +(lo) T EM/E (CCR7, CD45RA, CD57 ) T CM (CCR7+, CD45RA ) Other T cells T E/Exh (CCR7, CD45RA, CD57+) T EMRA (CCR7, CD45RA+) Nïve (CCR7+, CD45RA+, CD27+) b % CAR hi Medin EGFRt (FU) Among CAR hi Cells CD8 + Bulk-Derived T Cells Medin CD25 (FU) Among CAR hi Cells CD8 + T CM -derived Cells c CD8 + Bulk-Derived T Cells CD8 + T CM -derived Cells EGFRt CD25 d # Trget Cells Remining CD8 + T CM Cells Bulk CD8 + T Cells Fig. 1 (See legend on next pge.)

13 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 12 of 16 (See figure on previous pge.) Fig. 1 T CM -derived CAR-T cells produce more CAR hi T cells. Bulk T-cell derived CD4 + nd CD8 + CAR-T cells were co-incubted with CD19 + K562 trget cells without exogenous cytokines, nd T-cell subtype distribution ws quntified fter 24 h. T CM : centrl memory T cells; T EM : effector memory T cells; T EMRA : effector memory-cd45ra + T cells; T E : effector T cells; T Exh : exhusted T cells. b Bulk- nd T CM -derived CD8 + CAR-T cells were co-incubted with CD19 + K562 trget cells, nd the % CAR hi nd EGFRt nd CD25 expression levels mong CAR hi cells were quntified. c Both the bulk- nd T CM -derived popultions seprted into CAR hi /CD25 + nd CAR lo /CD25, with the T CM -derived smple showing higher CD25 expression overll. In both smples, subpopultion of contminting CAR cells ws lso CD25 +, possibly due to prcrine stimultion from CAR + cells. d Trget cell lysis ws monitored over 5 dys. Averge vlues of triplictes re shown with error brs indicting ± 1 s.d. stimulted with CD3/CD28 beds, lentivirlly trnsduced with CD19 CAR trnsgene, nd sorted for CAR + expression (Additionl file 13: Figure S13B). Without further expnsion, sorted CAR + cells were co-incubted with CD19 + K562 trget cells for 36 h. CAR hi /CAR lo bifurction ws observed in both T-cell popultions, but the T CM -derived popultion yielded more CAR hi cells, nd its CAR hi cells hd higher CAR nd CD25 expression levels (Fig. 1b). Consistent with ll previous experiments, which were performed using bulk CD8+ T cells, CAR lo cells from the bulk CD8 + -derived CAR-T smple were CD25.Incontrst, CAR lo cells from the T CM -derived smple hd prtilly upregulted CD25 (Fig. 1c), indicting tht the T CM -derived cells re both primed for the CAR hi phenotype nd ble to retin ctivtion sttus even when CAR expression levels re low. This conclusion is further supported by the observtion tht the T CM -derived cells were much more effective t lysing the trget cells (Fig. 1d). We monitored the T CM sttus of the two popultions throughout the cell preprtion process s well s fter ntigen stimultion. Surfce stining of CCR7 nd CD45RA reveled tht most cells in the T CM -enriched popultion hd lost CCR7 expression by the time of cell sorting for CAR expression (Additionl file 14: Figure S14A), progression tht is consistent with previous reports of ex vivo T CM cell expnsion [15, 16]. However, ntigen stimultion cused re-enrichment of the CCR7 + /CD45RA phenotype, prticulrly mong CAR hi cells (Additionl file 14: Figure S14B). In contrst, the bulk CD8 + Tcellpopultion enriched for CCR7 expression through the course of CD3/CD28 bed stimultion, nd further incresed CCR7 expression upon ntigen stimultion (Additionl file 14: Figure S14CD). Tken together, these results indicte the use of T CM cells s the strting popultion is conducive to the production of functionlly superior CAR-T cells, even though the dominnt phenotype remins fluid throughout the course of T-cell expnsion nd ntigen stimultion. Discussion Recent successes in CD19 CAR T-cell trils hve demonstrted the remrkble therpeutic potentil of CD19 CAR-T cells nd fueled intense interest in the development of CAR T-cell therpies ginst dditionl tumor trgets. As CAR T-cell technology moves beyond experimentl sttus, it is criticl tht effective cell-mnufcturing protocols nd chrcteriztion methods re developed to ensure robust nd reproducible genertion of CAR-T cells. The vrious clinicl trils completed thus fr hve employed different T-cell mnufcturing protocols unique to ech reserch group, nd the reltive merits of ech hve been judged bsed on overll clinicl outcome rther thn detiled exmintion of differences in T-cell chrcteristics. It is lso difficult to elucidte whether ny prticulr step in the preprtion process my hve ffected the phenotype nd functionlity of the resulting CAR-T cells. As result, efforts to improve cell preprtion protocols remin empiriclly driven, with few guiding cues on which prmeters to modify. Here, we presented systemtic study on the phenotypic nd functionl chnges of CAR-T cells fter T-cell ctivtion. We demonstrted tht ctivted T cells show cler pttern of popultion bifurction into CAR hi /CD25 + vs. CAR lo /CD25 groups. This bifurction ppers to be generl to T cells ctivted in vitro, s it is observed in both CD4 + nd CD8 + Tcells,inTcellsstimultedthrough either TCRs or CARs trgeting different ntigens, nd in T cells expressing CARs tht contin either CD28 or 4-1BB co-stimultory signls. The trnsient increse of CAR expression upon ntigen stimultion hs been observed in previous studies [29, 3], but to our knowledge, no detiled chrcteriztion of the difference between CAR hi nd CAR lo cells hs been performed. In this study, we discovered tht CAR hi cells consistently upregulte expression of the ctivtion mrker CD25, nd trget-cell lysis, cytokine production, nd T-cell prolifertion ssys demonstrte tht CAR hi /CD25 + cells re functionlly superior to their CAR lo /CD25 counterprts. In fct, CAR lo / CD25 cells show multiple signs of nergy nd re unble to execute nti-tumor functions. This observtion is of prcticl importnce becuse the CAR lo /CD25 popultion is CAR + with similr CAR genomic copy numbers s CAR hi cells; thus, they would hve stisfied the relese criteri typiclly pplied in CAR T-cell trils [4, 31] despite their lck of effector functions. The bility to distinguish nd chrcterize this popultion ws contingent upon the detiled in vitro exmintion of T-cell phenotype chnges post stimultion. Our results show tht the CAR hi /CD25 + phenotype is trnsient response to ntigen stimultion rther thn geneticlly hrd-wired popultion destined for superior

14 Chng et l. Journl of Trnsltionl Medicine (215) 13:161 Pge 13 of 16 function. Pst studies hve demonstrted tht T-cell ctivtion cn temporrily enhnce trnsgene expression from constitutive promoters, but hve not reveled the underlying mechnisms [32, 33]. A scn of the EF1α promoter used in the current study nd in multiple clinicl trils [31, 34, 35] revels binding sequences for TFII-I nd Sp1, both widely employed trnscription fctors. In prticulr, TFII-I hs been shown to be rpidly phosphorylted upon CD3 crosslinking nd upregulted in ctivted CD4 + T cells [36, 37]. It is possible tht T-cell signling increses the level of these trnscription fctors nd results in the upregultion of CAR expression from the EF1α promoter. Further investigtions re necessry to conclusively elucidte the mechnism of trnsient CAR upregultion upon ntigen stimultion. Although CAR hi /CD25 + cells eventully return to CAR lo /CD25 phenotype fter ntigen removl, they mount robust cytolytic nd cytokine-production response when re-chllenged with ntigen-expressing trgets. This is in strk contrst to CAR lo /CD25 cells, which remin non-functionl upon re-exposure to ntigen. Therefore, the bility to bis CAR-T cells towrd the CAR hi /CD25 + phenotype during the cell-preprtion stge hs the potentil to increse the therpeutic cpbility of T cells ginst trgeted tumor cells. We demonstrted tht, for second-genertion CAR contining CD28 co-stimultory domin, the ppliction of extr co-stimultion vi gonistic CD28 ntibody does not lter the reltive distribution of the CAR hi vs. CAR lo subpopultions, but sustined ntigen stimultion shows strong correltions with CAR hi cell genertion. Interestingly, the bsolute density of ntigen on trget cells did not correlte with the intensity of T-cell response or the effectiveness of CAR hi cell genertion. Among the ntigen-presenting cells tested, CD19 + K562 nd TM-LCL pper to present levels of ntigen stimultion tht re conducive to robust CAR hi /CD25 + cell production, consistent with TM-LCL s successful use in clinicl protocols [23, 38]. It remins possible tht chrcteristics in ddition to persistence, such s co-stimultory signls present on the trget cell surfce, contribute to the effectiveness of CD19 + K562 nd TM-LCL in supporting the genertion of CAR hi cells. Given the pprent sensitivity of CAR hi cell production to the type nd durtion of ntigen presenttion, nd the multiple degrees of vribility tht exist mong potentil feeder cell lines used in ex vivo T-cell expnsion, the detiled in vitro chrcteriztion pproch described in this study my be used to systemticlly fine-tune the ntigen-presenting cell type nd E:T rtio required for the efficient production of functionlly superior CAR-Tcells. In ddition to the type nd degree of ntigen stimultion used to expnd CAR-T cells, the specific T-cell subtype used to generte CAR-T cells lso gretly influences the efficiency of CAR hi cell genertion. Our study demonstrted tht CAR hi cells re enriched in the T CM phenotype, nd T CM -derived CAR-T cells re functionlly superior to those mde from bulk CD8 + T cells. These observtions re consistent with the previously reported observtion tht T CM cells re superior to T EM cells in estblishing long-term persistence in primtes [15]. In this nd most other CAR Tcell chrcteriztion studies, in vivo results re viewed s the most relevnt nd credible proof of CAR function. Although the vlue of in vivo dt is cler, mny importnt fetures of CAR-T cell biology prticulrly dynmic chnges over time in phenotype nd function re impossible to obtin t high enough resolution in vivo. Fetures such s CAR lo cells cnnot be detected in niml models or ptientsbecusetheyrequicklydepletedin vivo, butthe knowledge of their existence nd detiled in vitro chrcteriztions of such popultions provide vluble informtion on how to improve CAR-T cell production so s to mximize the number of cells tht will persist nd execute ntitumor functions upon doptive trnsfer. Recent clinicl studies hve demonstrted the exciting potentil of checkpoint inhibitor therpies such s CTLA-4 nd PD-1 blockde, which boost T-cell responses by preventing T-cell exhustion nd nergy. Our observtion tht the CAR hi phenotype is triggered by ntigen stimultion nd mintined by prolonged ntigen exposure rised the question of whether CAR hi cells my be susceptible to exhustion or dysfunction. We indeed observed significnt nd sustined PD-1 upregultion mong CAR hi cells. However, no functionl impirment ws observed even when CAR hi cells were chllenged over multiple dys with constnt supply of trget cells tht strongly overexpress PD-L1. It should be noted tht the K562 trget cells used in this study do not express the co-stimultory molecules CD83, CD86, 4-1BB lignd, OX4 lignd, nd ICOS lignd, nd they express very low levels of CD8 [39]. Pst studies hve needed to engineer incresed expression of these co-stimultory molecules on K562s in order to prime nd expnd T cells [39 41]. Therefore, the K562 cells used in our study, which hve not been engineered to express co-stimultory molecules, re unlikely to significntly mitigte PD-1/PD-L1 signling vi CAR-independent co-stimultion. Our results contrst with reports of PD-L1 sensitivity exhibited by T cells stimulted vi nturl TCRs [42 44], nd indicte n unexpected source of resilience in CAR-T cells ginst PD-1 medited cell inctivtion. Intensive T-cell stimultion with CD3 nd CD28 ntibodies hs been found to overcome PD-1/ PD-L1 inhibition of T-cell prolifertion in vitro [45]; it is possible tht robust expression nd ctivtion of the exogenous CAR contining CD28 co-stimultory domin similrly overwhelms PD-1 signling. However, PD-L1 blockde hs been reported to restore ctivity in hypofunctionl CAR-T cells ex vivo fter recovery from