2054 haematologica | 2021; 106(8)
Received: December 7, 2020.
Accepted: March 11, 2021.
Pre-published: April 1, 2021.
©2021 Ferrata Storti FoundationMaterial published in Haematologica is covered by copyright.All rights are reserved to the Ferrata Storti Foundation. Use ofpublished material is allowed under the following terms andconditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. Copies of published material are allowed for personal or inter-nal use. Sharing published material for non-commercial pur-poses is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode,sect. 3. Reproducing and sharing published material for com-mercial purposes is not allowed without permission in writingfrom the publisher.
Correspondence: BENEDETTO [email protected]
Haematologica 2021Volume 106(8):2054-2065
REVIEW ARTICLE
https://doi.org/10.3324/haematol.2020.276402
Ferrata Storti Foundation
Chimeric antigen receptor (CAR) T cells (CAR-T) have dramaticallychanged the treatment landscape of B-cell malignancies, providinga potential cure for relapsed/refractory patients. Long-termresponses in patients with acute lymphoblastic leukemia and nonHodgkin lymphomas have encouraged further development in myeloma.In particular, B-cell maturation antigen (BCMA)-targeted CAR-T haveestablished very promising results in heavily pre-treated patients.Moreover, CAR-T targeting other antigens (i.e., SLAMF7 and CD44v6) arecurrently under investigation. However, none of these current autologoustherapies have been approved, and despite high overall response ratesacross studies, main issues such as long-term outcome, toxicities, treat-ment resistance, and management of complications limit as yet theirwidespread use. Here, we critically review the most important pre-clinicaland clinical findings, recent advances in CAR-T against myeloma, as wellas discoveries in the biology of a still incurable disease, that, all together,will further improve safety and efficacy in relapsed/refractory patients,urgently in need of novel treatment options.
European Myeloma Network perspective onCAR T-cell therapies for multiple myelomaBenedetto Bruno,1,2º Ralph Wäsch,3 Monika Engelhardt,3 Francesca Gay,1Luisa Giaccone,1 Mattia D’Agostino,1 Luis-Gerardo Rodríguez-Lobato,4,5 Sophia Danhof,5 Nico Gagelmann,6 Nicolaus Kröger,6 Rakesh Popat,7Niels W C J van de Donk,8 Evangelos Terpos,9 Meletios A Dimopoulos,9Pieter Sonneveld,10 Hermann Einsele5 and Mario Boccadoro11Department of Molecular Biotechnology and Health Sciences, University of Torino andDepartment of Oncology, Division of Hematology, A.O.U. Città della Salute e della Scienzadi Torino, Presidio Molinette, Torino, Italy; 2Division of Hematology and Medical Oncology,Perlmutter Cancer Center, Grossman School of Medicine, NYU Langone Health, New York,NY, USA; ; 3Department of Hematology, Oncology and Stem Cell Transplantation, UniversityMedical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany; 4Unit ofAmyloidosis and Multiple Myeloma, Department of Hematology, Hospital Clínic ofBarcelona, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona,Spain; 5Division of Medicine II, University Hospital Würzburg, Würzburg, Germany;6Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf,Hamburg, Germany; 7Department of Hematology, University College London Hospitals,London, UK; 8Department of Hematology, Amsterdam University Medical Centers, CancerCenter Amsterdam, Location VUmc, Amsterdam, the Netherlands; 9Stem CellTransplantation Unit, Plasma Cell Dyscrasias Unit, Department of Clinical Therapeutics,School of Medicine, National and Kapodistrian University of Athens, Athens, Greece and10Erasmus MC Cancer Institute, Rotterdam, the Netherlands.
ºCurrent address: Division of Hematology and Medical Oncology, Perlmutter Cancer Center,Grossman School of Medicine, NYU Langone Health, New York, NY, USA.
ABSTRACT
Introduction
Recently engineered chimeric antigen receptors (CAR) have greatly increasedanti-tumor effects of CAR T cells (CAR-T). Impressive results have been observedwith CD19-directed CAR-T in B-cell lymphoproliferative disorders.1-3 In addition,several CAR-T products have been developed for the treatment of multiple myelo-ma (MM). None has yet been approved, and, despite high overall response (OR)across studies, main issues such as long-term outcomes, toxicities and complica-tions need to be solved to allow their widespread clinical use. In this review, theEuropean Myeloma Network (EMN) group aimed to describe the most importantpre-clinical and clinical findings, and recent advances in CAR-T technology againstMM that may improve their safety and efficacy. Contents, comments and sugges-
tions have been incorporated in the manuscript after atleast three rounds of discussion resulting in the final unan-imously approved version.
Target antigens
CAR are artificial fusion proteins with a modular designthat confer antigen-specificity to T cells in an humanleukocyte antigen (HLA)-independent manner providingintracellular stimulatory signals to enhance survival, pro-liferation, cytolytic capacity and cytokine production of Tcells.4 Figure 1 illustrates the components of CAR con-structs.5-8 For successful CAR-T therapy, identification ofsuitable tumor antigens is crucial, since it requires a deli-cate balance between effectiveness and safety considera-tions. Ideal antigens should be: (i) highly and homoge-neously expressed on tumor cell surface, (ii) expressed atdifferent disease stages, (iii) pivotal in disease pathophys-iology, (iv) limited or not shed into the bloodstream, (v)not affected by selective treatment pressure that maycause down-regulation or elimination, and (vi) not
expressed on normal tissues.9-10 Great progress has beenmade to identify potential molecules as CAR targets inMM. In this section, we summarize pre-clinical data onthe most relevant MM-associated antigens, while a com-prehensive overview is provided in Table 1.
B-cell maturation antigen
The B-cell maturation antigen (BCMA) gene is locatedon chromosome 16 and the BCMA (aliases: CD269,TNFRSF17) protein, a transmembrane glycoprotein mem-ber of the tumor necrosis factor receptor (TNFR) super-family, is expressed on subsets of B cells (plasmablasts andplasma cells) and up-regulated during B-cell differentia-tion. It is not expressed on solid organ tissues, hematopoi-etic cells or naïve B cells.11-12 Along with two associatedreceptors (calcium modulator and cyclophilin ligand inter-actor [TACI] and B-cell activation factor receptor [BAFF-R]) and its ligands (a proliferation inducing ligand [APRIL]and B-cell activating factor [BAFF]) BMCA regulates matu-ration, differentiation, and promotes B-cell survival.13-15
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Figure 1. Chimeric antigen receptor T cells. Chimeric antigen receptors (CAR) are designer proteins that redirect T cells towards a defined surface antigen on tumorcells. The CAR construct contains four essential components. The extracellular antigen recognition domain consists of a single chain variable fragment (scFv) com-monly derived from the variable domains of the heavy and light chains (VH and VL) of monoclonal antibodies joined by a linker to provide flexibility and solubility andtherefore improve antigen recognition and binding capacity. The hinge or spacer moiety based on Ig- (IgG1 or IgG4), CD8- or CD28-derived domains, provides flexi-bility, stability and the suitable length for optimal access to the target antigen. The transmembrane domain links the extracellular and intracellular domains of theCAR. It is based on CD3!, CD4, CD8", CD28 or ICOS moieties, influences CAR stability and signaling and may also be involved in immune synapse arrangement. Thelast components of the CAR construct are the intracellular signaling domains. The activation domain is typically derived from the CD3! moiety of the T-cell receptor(first generation CAR), whereas co-stimulatory domains are derived from CD28, 4-1BB, OX40, CD27, or ICOS (second and third generation CAR). Co-stimulationresults in intracellular signals that further optimize T-cell function, persistence and proliferation. Through additional genetic modifications, so called “armored” CART cells (CAR-T) (fourth generation CAR) secrete cytokines or express ligands to bolster CAR-T function or to overcome the immunosuppressive tumor microenviron-ment. Taken together, the molecular fine-tuning of pre-existing CAR components can greatly improve cellular migration, foster expansion and persistence of the CAR-T and decrease toxicity.
Expression of BCMA in malignant plasma cells isenhanced compared to non-malignant cells, though levelsare not homogeneous. Its expression is associated withproliferation and survival of tumor cells and contributes tothe immunosuppressive bone marrow (BM) microenvi-ronment.15-17 BCMA cleavage by #-secretase sheds solubleBCMA (sBCMA) into the bloodstream.18 sBCMA mayplay a role in myeloma pathogenesis, and high sBCMAlevels have been associated to worse prognosis.19 BCMA iscurrently considered the most compelling antigen for tar-geted immunotherapy. Carpenter et al. reported on thefirst proof-of-concept using a second generation, CD28co-stimulated CAR against BCMA in the preclinical set-ting. BCMA CAR-T specifically recognized the antigen,eradicated in vivo tumors and killed primary myelomacells,11 setting the cornerstone for the first-in-human phaseI clinical trial evaluating BCMA CAR-T in MM.20
Transmembrane activator, calcium modulator,and cyclophilin ligand interactor
TACI (TNFRSF13B) is a transmembrane protein that
recognizes ligands APRIL, BAFF and calcium modulatorand cyclophilin ligand (CAML). It is expressed on subsetsof naïve and memory B cells, plasma cells, non-germinalcenter cells, monocytes and dendritic cells. TACI supportsgrowth and survival in myeloma cells, though its expres-sion is lower compared to BCMA.21-23 A third-generationAPRIL-based CAR recognizing both BCMA and TACIantigens has been engineered. Though this constructdemonstrated tumor control in an in vivo model of tumorescape with BCMA- TACI+ cells,22 the AUTO2 trial (clini-caltrials gov. Identifier: NCT03287804) was, however ter-minated because of lack of efficacy.24
CD19In most B-cell malignancies, CD19 is highly and uni-
formly expressed.25-29 MM was traditionally consideredmostly CD19 negative with low level CD19 expressionattributed to a putative “myeloma stem cell”. However,highly sensitive direct stochastic optical reconstructionmicroscopy (dSTORM) unveiled expression of CD19 on aconsiderable subset (10–80%) of myeloma cells in morethan two thirds of patients, of whom only one fifth wasconsidered CD19 positive by conventional flow cytome-
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Table 1. Potential target antigens for CAR-T therapy for mutiple myeloma.Antigen Expression in MM Expression in normal Expression in healthy solid Development state hematopoietic cells organ tissues
BCMA 60-100% Late memory B cells, plasma cells No Clinical trialTACI 78% Naïve and memory B cells, plasma No Clinical trial cells, monocytes and dendritic cellsCD19 10-80% B-cells, plasma cells No Clinical trialSLAMF7 (CD319) High and uniform expression NK-cells, monocytes, macrophages, No Clinical trial dendritic cells, T cells, B cells, plasma cells CD38 High and uniform expression Lymphoid and myeloid cells, Prostatic epithelium, Clinical trial hematopoietic precursors, pancreatic islet cells, thymocytes cerebellar Purkinje cells CD44v6 43% in advanced stage Activated T cells, monocytes Keratinocytes Clinical trialGPRC5D $50% in 65% of patients B-cells, plasma cells Hair follicles Clinical trialCD138 High expression Plasma cells Epithelial cells, gastrointestinal Clinical trial tract and hepatocytesNKG2D Heterogenous NK, T and #% T cells No Clinical trial& light chain !-restricted myeloma cells Mature B cells No Clinical trialCD56 High expression, decreased T and NK cells Central and peripheral Clinical trial in extramedullary disease nervous systemLewis Y 50% No Epithelial cells Clinical trialNY-ESO-1 60-100% No No Clinical trialCD229 (SLAMF3) High and homogeneous T, NK and B cells No Preclinical investigation expression, probably in myeloma stem cell Integrin '7 High expression High expression in B cells and No Preclinical investigation low to moderate expression in CD34+ hematopoietic cellsCD70 0.2-42% Activated T and B cells, dendritic No Preclinical investigation cells and plasma cellsCD1d High expression Antigen-presenting cells, Epithelial cells Preclinical investigation thymocytes, B cells, and hematopoietic stem cells
BCMA: B-cell maturation antigen; GPRC5D: G protein-coupled receptor class C group 5 member D; NKG2D: Natural Killer Group 2 member D; NY-ESO-1: New York Esophageal SquamousCell Carcinoma 1; SLAMF3 and SLAMF7: signaling lymphocytic activation molecules family member 3 and 7; TACI: Transmembrane activator, calcium modulator, and cyclophilin ligandinteractor.
try. As CAR-T can eliminate cells expressing less than 100target antigens/cell, CD19 has become a relevant CAR tar-get antigen. In preclinical models, BCMA-CD19 bispecificCAR-T eliminated myeloma cell lines more potently thanBCMA- or CD19-directed CAR-T alone.30 Due to an off-target expression limited to B cells, toxicity concerns of(co-)targeting CD19 are limited and clinical evaluation ofbispecific CAR-T is ongoing (clinicaltrials gov. Identifier:NCT03455972, NCT03549442).
SLAMF7The elotuzumab target antigen signaling lymphocytic
activation molecule (SLAM) family member 7 (SLAMF7,aliases: CD319, CS-1, CRACC) is an immunomodulatorytransmembrane receptor, initially identified on the surfaceof natural killer (NK) cells.31 It is expressed on a variety ofother innate immune cells,32 but also T cells, B cells andplasma cells.31-33 Importantly, SLAMF7 is expressed onaberrant plasma cells and its precursor34 and confers hom-ing of the myeloma cells to the BM niche. While redirect-ing T cells against a self-antigen may appear difficult, pre-clinical experiments demonstrated that it is feasible togenerate clinically relevant doses of SLAMF7-directedCAR-T, with or without additional inactivation of theendogenous SLAMF7 gene.33-35 In preclinical models,potent anti-myeloma activity was demonstrated, resultingin rapid, comprehensive and sustained cell depletion.33SLAMF7-directed CAR-T eliminated SLAMF7 positivelymphocytes in vitro, while SLAMF7 negative lympho-cytes were spared and retained their functions.33 Clinicalevaluation of SLAMF7 CAR-T with functional safetyswitches is currently ongoing (clinicaltrials gov. Identifier:NCT03958656, EudraCT Nr.2019-001264-30).
CD38Successfully targeting CD38 (cyclic ADP ribose hydro-
lase, ADPRC1) with daratumumab and isatuximab has ledto the development of anti-CD38 CAR-T. CD38 is a trans-membrane glycoprotein that functions as an ectoenzyme,adhesion molecule and regulator of migration and signal-ing. It is expressed on malignant plasma cells,36 but lowexpression can be found on lymphoid and myeloid cells,hematopoietic precursors,37 thymocytes, cerebellarPurkinje cells and other tissues. CD38 is an activationmarker of T cells at intermediate or late activation stages.As CD38-directed CAR-T demonstrated great antigen-specific efficacy in preclinical myeloma models,38 affinitymodification of the CAR was developed as an approach tomitigate on-target, off-tumor toxicity towards other CD38positive hematopoietic cells. Affinity reduction of theantigen binding domain by a factor of 1,000 enabled selec-tive elimination of myeloma cells with high CD38 expres-sion while sparing normal cells with less pronouncedCD38 expression. However, it has been reported that lev-els of CD38 expression on myeloma cells can decline overthe disease course.39 In this regard, agents that induceselective modulation of CD38 expression levels, such asall-trans retinoic acid (ATRA) or histone deacetylase(HDAC) inhibitors,40 represent a promising group for com-bination therapy with CD38-directed CAR-T. In order toaddress the issue of antigen reduction by increasing thepotency of the cell product, a novel construct termed“dimeric antigen receptor” (DAR) was developed. In fact,the DAR T cells that incorporate a fragment antigen-bind-
ing (Fab) moiety instead of the single chain variable frag-ment (scFv), demonstrated superior preclinical activity.However, their clinical relevance, and the risk of on-target,off-tumor effects, remains to be determined.
CD44v6CD44 glycoproteins are encoded by a highly conserved
gene,41 but are nevertheless characterized by considerableprotein heterogeneity due to post-transcriptional modifi-cations or splicing variants. While CD44 plays a role inphysiological processes and is expressed on healthy tis-sues,42 the isoform variant 6 is relatively restricted tomalignant cells43,44 including plasma cells. In healthy tis-sues, CD44v6 expression is limited to skin keratinocytes.It is absent on hematopoietic precursor cells, but low levelexpression can be found on activated T cells and mono-cytes. While the development of an anti-CD44v6immunoconjugate was discontinued due to severe skintoxicities,45 preclinical investigation of CD44v6-directedCAR-T showed promising efficacy with no impact on ker-atinocytes that represent potentially immune-privilegedsites.46 The clinical relevance of the observed monocytedepletion remains unclear. However, as monocyte-derivedcytokines play a relevant role for the pathogenesis ofcytokine release syndrome (CRS) and immune effectorcell-associated neurotoxicity syndrome (ICANS), a benefi-cial effect is possible and clinical evaluation is ongoing(clinicaltrials gov. Identifier: NCT04097301).
GPRC5D The orphan G protein–coupled receptor, class C group 5
member D (GPRC5D), is expressed ubiquitously in malig-nant bone marrow plasma cells (500 to 1,000 times higherexpression than on normal cells), hair follicles, and in thelung. CAR-T targeting GPRC5D have demonstratedpromising preclinical activity, and a phase I clinical trial isongoing (clinicaltrials gov. Identifier: NCT04555551).
CD229 CD229, a SLAM family receptor ("SALM3"), is generally
expressed on myeloma cells and "precursor" myelomacells. Its high expression suggests a potential as a target forCAR-T studies have shown that this newly designedCD229 CAR-T has high activity against MM cells, mem-ory B cells and MM stem cells in vitro and in vivo.47
Clinical studies
B-cell maturation antigen-specific chimeric antigenreceptor T cells
The National Cancer Institute group performed the firststudy with BCMA-specific CAR-T with a CD28 costimu-latory domain (murine scFv) in heavily pretreatedpatients.20 At the highest CAR-T dose (9×106 cells/kg), 13of 16 patients (81%) achieved at least partial response (PR)with a median event-free survival of 31 weeks. Otherstudies confirmed high activity of BCMA CAR-T in thispatient subset.48-50 Advanced clinical findings have beenreported with ide-cel50,51 and cilta-cel.48 Both therapiesreceived Food and Drug Administration breakthroughdesignation. In this section, we will discuss these twoCAR-T products. CAR-T constructs and main clinicalcharacteristics are summarized in Table 2.
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Ide-cel (bb2121)The first-in-man study with ide-cel (CRB-401) evaluated
escalating doses of CAR-T (50×106, 150×106, 450×106, or800x106 in the dose-escalation phase, and 150×106-450x106in the expansion phase) in extensively pretreated MM(median of six prior therapy lines; 69% triple-class refrac-tory).51 Sixty-two patients were enrolled. At least PR wasachieved by 76% of patients including complete response(CR) in 39%. All 15 patients with $CR who had an assess-ment for minimal residual disease (MRD) were MRD-neg-ative at the level of 10-5. Baseline BCMA expression orsBMCA levels did not affect response. There was a trendtowards lower response in patients who received
(150×106 CAR-T, in those with less in vivo CAR-T expan-sion, and in those with high-risk cytogenetic abnormali-ties. Median progression free survival (PFS) was 8.8months for all patients, and 9.0 months for those whoreceived 450×106 CAR-T. Median overall survival (OS)was 34.2 months. Based on these promising results of thephase I trial, a second trial (KarMMa, phase II study) wasinitiated to evaluate the value of ide-cel in larger numbersof patients who were previously exposed to immunomod-ulatory drugs (IMiD), a proteasome inhibitor, and a CD38antibody.50 In this study 140 patients were enrolled with amanufacturing success of 99%; 128 of 140 (91%) receivedCAR-T, whereby 88% received bridging therapy prior to
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Table 2. Ide-cel and Cilta-cel: CAR-T and clinical characteristics Ide-cel (bb2121) / KarMMa (phase II) Cilta-cel (JNJ-4528) / CARTITUDE-1 (phase IB/ II)
Antigen-binding domain scFv (murine) Bispecific variable fragments of llama heavy-chain antibodies; two distinct BCMA epitopes are targetedSignaling domains CD3!/4-1BB CD3!/4-1BBVector Lentiviral Lentiviral Other features Bb21217 uses the same CAR construct as used for ide-cel. Bi-epitope BCMA binding confers high avidity binding During ex vivo culture a PI3K inhibitor is added to enrich for CAR-T with memory-like phenotype Lymphodepletion Flu/Cy Flu/CyCAR-T dose 150-450×106 Median dose: 0.71×106/kgNumber of patients 128 (140 patients underwent leukapheresis) Data presented for first 97 (113 patients were enrolled/apheresedBridging therapy (%) 88 65Number of prior therapies (median) 6 6Triple-class refractory (%) 84 88High-risk cytogenetics (del(17p), t(4;14), or t(14;16) (%) 35 24Extramedullary disease (%) 39 13$PR 150-450×106: 73% 97% 150×106 : 50% 300×106 : 69% 450×106 : 82%$CR 150-450×106: 33% 67% 150×106 : 25% 300×106 : 29% 450×106 : 39%Median PFS 150-450×106: 8.8 months Median PFS: Not reached; 12-month PFS rate: 77% 150×106 : 2.8 months 300×106 : 5.8 months 450×106 : 12.1 monthsCRS (all grades) (%) 84 95CRS (grade $3) (%) 5 4Median time to CRS onset 1 7
(any grade) (days)Median duration of CRS
(any grade) (days) 5 4Neurotoxicity (all grades) (%) 18 21 (ICANS: 17%; other neurotoxicity*: 12%)Neurotoxicity (grade $3) (%) 3 10 (ICANS: 2%; other neurotoxicity: 9%)Median time to neurotoxicity onset
(any grade) (days) 2 ICANS: 8 days; other neurotoxicities*: 27 daysMedian duration of neurotoxicity
(any grade) (days) 3 ICANS: 4 days; other neurotoxicities: 75 daysTime to peak CAR-T expansion (days) 11 13CAR-T persistence 6 months, % 59 42
*Other neurotoxicities are defined as neurotoxicities occurring after resolution of cytokine release syndrome (CRS) and/or immune effector cell-associated neurotoxicity syndrome(ICANS). PR: partial response; CR: complete response; PFS: progression free survival.
CAR-T. Patients were highly pretreated with a median ofsix prior therapy lines and 84% had triple-class refractorydisease (refractory to one protease inhibitor [PI], oneIMiD, and a CD38 antibody). At least PR was achieved by73% including $CR in 33%. MRD-negative CR wasachieved in 26%. Median time to response was 1 month.Fifty-four patients, who received 450×106 CAR-T, hadsuperior response ($PR: 82%; $CR: 39%; MRD-negativeCR: 28%) when compared to lower doses. RevisedMultiple Myeloma International Staging System (R-ISS)stage 3 disease at enrollment had inferior response, com-pared to R-ISS stage 1 or 2. As in the phase I trial, baselineBCMA expression did not affect response to ide-cel. Withmedian follow-up of 13.3 months, overall median PFS was8.8 months. Median PFS increased with higher CAR-Tdose with a median PFS of 12.1 months for patients whoreceived 450×106 CAR-T. Patients, who achieved at leastCR, also experienced better PFS ($CR: median PFS of 20.2months; very good partial response [VGPR]: median PFSof 11.3 months; PR: median PFS of 5.4 months; no-response: 1.8 months). Median OS was 19.4 months.Durable CAR-T persistence was observed up to 1 year:CAR-T were detected at 1, 3, 6, 9, and 12 months in 99%,75%, 59%, 37%, and 46% respectively. CAR-T expansionwas increased at higher doses. In an ongoing phase IIIstudy, ide-cel is compared with standard-of-care regimensin patients with 2-4 prior regimens, including IMiD, PI,and CD38 antibody (KarMMa-3). Ide-cel is also evaluatedin the multi-cohort KarMMa-2 study, in patients withearly relapse after first-line therapy or patients with sub-optimal response after autografting (<VGPR).
Cilta-cel (JNJ-4528)The CARTITUDE-1 study evaluates cilta-cel (target
dose: 0.75×106 CAR+ T /kg) in patients exposed to PI,IMiD and CD38 antibody. Preliminary results were pre-sented at the 2020 ASH conference. Sixteen of 113patients, who underwent apheresis, were not dosedbecause of consent withdrawal (n=5), progressive disease(n=2) or death (n=9). The remaining 97 patients hadreceived a median of six prior lines of therapy. Ninety-seven percent of patients achieved at least PR with strin-gent CR in 67%. Fourty-eight of the 57 patients evaluablefor MRD, 93% were MRD-negative at the level of 10-5.Response was independent of baseline BCMA expression.Median time to first response was 1 month. At a medianfollow-up of 12.4 months, 12-month PFS was 77%. PeakCAR-T expansion was observed around day 10-14, andCAR-T were observed in 36% of patients at 3 months offollow-up.52 Interestingly, response to cilta-cel was inde-pendent of CAR-T expansion and persistence.52 In theChinese LEGEND-2 trial, different conditioning regimenswere used, as well as variable CAR-T infusion methods(split vs. single infusion). The Xi’an site, which usedcyclophosphamide lymphodepletion therapy and threeCAR-T cell infusions (dose: 0.07-2.1×106/kg; median dose:0.50×106/kg), enrolled 57 out of 74 patients.53 Thesepatients had received a median of three prior lines of ther-apy (prior PI and IMiD: 60%). Overall response rate (ORR)was 88% with CR in 74% (median time to response: 1month). MRD-negative CR was achieved in 68%. At amedian follow-up of 25 months, median PFS was 19.9months for all patients, while it was 28.2 months for thosein CR. Median OS was 36.1 months (not reached forpatients in CR). Cilta-cel is also being evaluated in a phase
III study (CARTITUDE-4), which compares CAR-T versuspomalidomide, bortezomib and dexamethasone or dara-tumumab, pomalidomide and dexamethasone in relapsedand lenalidomide-refractory MM. In addition, the ongoingCARTITUDE-2 study is evaluating cilta-cel in differentpatient populations, including those with early relapseafter frontline therapy, prior exposure to a BCMA-target-ing drug, and those with <CR post-auto-SCT.
Other B-cell maturation antigen-specific chimeric antigen receptor T cells
In order to further improve the activity and/or persist-ence of CAR-T, several studies are evaluating novelBCMA-targeting CAR-T. Studies include CAR constructscontaining a fully human BCMA-specific binding domainto reduce development of humoral and/or cellularimmune responses against CAR-T, which may impairCAR-T persistence.49,54-56 One of these products with afully human antigen-binding domain is orva-cel (orvacab-tagene autoleucel), which is currently evaluated in thephase I/II EVOLVE study. This study shows promisingefficacy of orva-cel in heavily pretreated MM (median ofsix prior lines of therapy; 94% triple-class refractory). Atleast PR was achieved in 92% of 62 patients treated athigher dose levels (300-600×106 CAR-T), with CR in 36%.Follow-up is ongoing. Treatment was associated with alow incidence of grade $3 cytokine release syndrome (3%)and grade $3 neurotoxicity (3%). Following CAR-T, therewas robust expansion and durable persistence (69% ofpatients had detectable CAR-T at 6 months). Moreover,preclinical studies have shown that enrichment forBCMA-targeting CAR-T displaying a memory-like pheno-type leads to improved persistence in mouse models,57which may result in more durable disease control.Bb21217 uses the same CAR molecule as ide-cel, butbb21217 is cultured in the presence of a PI3 kinaseinhibitor, which leads to enrichment for CAR-T with amemory-like phenotype. Preliminary results showed effi-cacy in 69 heavily pretreated patients (64% triple-classrefractory) with an ORR of 68% (CR of 29%; medianresponse duration: 17.0 months). Interestingly, a highmemory-like T-cell count in the drug product was associ-ated with superior CAR-T expansion and less progressionat 6 months. The manufacturing process for orva-cel isalso designed to produce CAR-T enriched for centralmemory T-cell phenotype.56 Other trials are evaluatingcombinations of CAR-T with other drugs to improveactivity and durability of response. Based on preclinicaldata showing that the T-cell stimulatory effects of IMiDenhance the efficacy of CAR-T,58-60 several ongoing clinicalstudies are evaluating the combination of lenalidomideand CAR-T. The combination of BCMA CAR-T with #-secretase inhibitors is also investigated, because in vitrostudies show that #-secretase inhibitors block BCMAcleavage and increase BCMA cell surface expression lev-els.54,61 These results are confirmed in an ongoing clinicalstudy, which shows that gamma secretase inhibitionenhances BCMA surface expression on MM cells andreduces soluble BCMA levels.54
CD19-specific CAR-T Recent studies showed that MM cells express ultra-low
levels of CD19,30 moreover MM cells with disease-propa-gating properties also express CD19. This formed therationale for the evaluation of CD19-specific CAR-T in
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MM.62 One study evaluated CD19 CAR-T following sal-vage high-dose therapy and autologous transplantation.All ten patients had shown a PFS shorter than 1 year froma previous transplant. Two experienced a longer PFS aftertransplant plus CD19 CAR-T therapy, compared with firstautologous stem-cell transplantation. Only one patientexperienced CRS (grade 1).63
Chimeric antigen receptor T-cell toxicity in multiple myeloma
CAR-T may induce several life-threatening side effectssuch as CRS and ICANS.64 Hemophagocytosis and pro-longed cytopenias may also occur. CRS mainly consists offever, hypotension, hypoxia and organ toxicity, whichmay result in organ failure. ICANS may include severalsymptoms: impaired concentration, cognitive disorders,confusion, agitation, tremor, lethargy, aphasia, delirium,somnolence, seizures, motor weakness, paresis or signs ofintracerebral pressure. ICANS usually occurs during orafter CRS and may manifest a biphasic course, in about10% of cases up to 4 weeks after CAR-T infusion. A ten-point neurologic assessment, at least twice a day, using theICE screening tool is recommended for early detection.64Reported toxicity rates of hallmark studies are illustratedin Table 3. Importantly, a broad consensus statementoffering updated comprehensive recommendations for thetreatment of toxicities associated with immunotherapieshas been recently published.65 Though many aspectsremain unknown, mechanisms underlying CRS andICANS have become clearer. Several factors contribute todifferent toxicity rates.65 Moreover, incidence and severityof adverse effects vary between diseases. While the inci-dence of any grade CRS is comparable between diseases,CRS severity ($grade 3) is highest in patients with ALLand lowest in MM. This may partly be explained by dis-ease burden and aggressiveness. In addition, earlier treat-ment of CAR-T side effects with more experience in
recent trials may have contributed to reduce progressionto higher grades of toxicity. Unlike CRS, incidence ofICANS is higher in ALL or lymphoma and appears lowerin MM patients. Other factors such as tumor burden, priortreatment, CAR-T constructs and dose administered havebeen described. Several grading systems have been pro-posed to assess CRS and ICANS. Recently, the AmericanSociety for Transplantation and Cellular Therapy gradingsystem was compared to other grading scores in two adultpopulations.66 Interestingly, incidence of CRS and ICANSwere similar in all grading systems. By contrast, only 25%and 54% of patients were however assigned to the sameseverity grade given the discrepancies in scoring adversesymptoms. These differences may also easily lead toinconsistent management guidelines among studies.Overall, efforts should be made to unify grading systemsbe used across clinical trials.
Improving chimeric antigen receptor T-cell ther-apies in multiple myeloma
Despite numerous autologous CAR-T products underdevelopment67,68 and encouraging high response rates,69none have yet been approved, with BCMA remaining thebest evaluated target.49,51,69 Other limitations are toxicities,resistance mechanisms, availability, and patient manage-ment (Table 4). Here we highlight possible strategies forimprovement.70,71
Safety Preventing cytokine release
The pro-inflammatory interleukin-6 (IL-6) is increasing-ly acknowledged to play a central role in the pathogenesisof CRS.47 A recent study designed a nonsignaling mem-brane-bound IL-6 receptor (mbaIL6) which was constitut-ed by a scFv derived from an antibody against IL-6, andlinked to a transmembrane anchoring peptide. The studyidentified expression of mbaIL6 on the surface of T cells
Table 3. Toxicity of CAR-T cell treatment in multiple myelomaCAR-T Construct Cell dose Trial Sponsor N Cytopenia 3/4 CRS 3/4 ICANS 3/4 Ref
BCMA/CD28 9×106 cells / kg bw First-in-humans NIH 16 leucopenia 94% 38% 6% 30 neutropenia 88% thrombopenia 63% prolonged 13%BCMA/4-1BB C1: 1-5×108 total cells Phase I UPenn 25 leucopenia 44% 32% 12% 49 C2: Cy+1-5×107 total cells neutropenia 44% C3: Cy+1-5×108 total cells thrombopenia 28% prolonged n.r.BCMA/4-1BB LCAR-B38M med. 0.5×106 cells / kg bw LEGEND-2 China 57 leucopenia 30% 7% 0% 53 (JNJ-4528) (Phase 1) neutropenia n.r. thrombopenia 23% prolonged 16% BCMA/4-1BB JNJ-4528 med. 0.73×106 cells / kg bw CARTITUDE-1 Janssen 29 leucopenia 59% 7% 3% 48 (Phase I/II) neutropenia 100% 68 thrombopenia 69% prolonged n.r.BCMA/CD28 bb2121 50-800 x 106 total cells CRB-401 BMS / Celgene 33 leucopenia 58% 6% 3% 51 (Phase I) neutropenia 85% thrombopenia 45% prolonged n 3% t 35%CRS: cytokine-release syndrome; ICANS: immune effector cell-associated neurotoxicity syndrome; Ref.: references; bw: body weight; C: cohort; Cy: cyclophosphamide; n.r.: not reported; n: number of patients.
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which was associated with rapid removal of IL-6 from theculture supernatant. Furthermore, T-cell proliferationincreased while the signaling and function of IL-6 wasneutralized. T cells co-expressing mbaIL6 and anti-CD19CAR, neutralized IL-6 derived from macrophages whilemaintaining antitumor activity in vitro and in a xenograftmodel.72 Thus, CAR-T incorporating the capacity toremove IL-6 may provide new strategies for preventingCRS. Another strategy is the incorporation of “suicide”switches, such as constructs containing a CAR andinducible caspase 9. Administration of a small moleculecausing dimerization of inducible caspase 9 resulted inapoptosis and CAR-T-specific depletion.73
Reducing off-tumor on-target toxicity In order to avoid adverse off-tumor effects, spatial and
temporal activity of CAR-T need to be limited. Under thishypothesis, GPRC5D has been proposed as a novel targetantigen, expressed on almost all CD138+ cells.74 LikeBCMA, its expression is restricted to plasma cells, exceptfor hair follicles. Preliminary results of anti-GPRC5DCAR-T showed potent anti-MM efficacy in vitro and in amouse model, with the encouraging finding that thesecells also effectively eradicated MM after treatment withanti-BCMA CAR-T. Most recently, it was demonstratedthat simultaneous targeting of GPRC5D and BCMA couldprevent relapse mediated by BCMA escape. Several multi-target constructs were compared and in BCMA-negativedisease, dual-target (bicistronic) and pooled approachesexhibited the highest efficacy, whereas forGPRC5D/BCMA-expressing disease, the dual-targetappeared to be more efficacious. Mechanistically, express-ing two CAR on one cell enhanced the strength of CAR-T/target interactions.
Reducing immunogenicity and simplifying structures In order to reduce the immunogenicity of the CAR bind-
ing domain, human or humanized scFv have been usedmore frequently in recent studies, instead of murinesequences.68,75 Furthermore, a reduction of immunogenici-
ty might be achieved by the incorporation of heavy-chain-only binding domains, which subsequently simplify thestructure of the CAR antigen-binding domain withouthaving a light-chain domain.76 In general, simplified struc-tures may facilitate better gene expression by transducedT cells.76,77 Moreover, limiting the size of expressed genesis important for the potential expression of >1 protein.78,79A recent study demonstrated that CAR with antigen-recognition domains consisting of only a fully humanheavy-chain variable domain (FHVH33) in addition to 4-1BB and CD3! domains mediated comparable cytokinerelease, reduction in tumor burden, and degranulation inmice when compared to an identical CAR with a conven-tional scFv.76,80 Further investigations identified a crucialcontribution of 4-1BB in reducing activation-induced celldeath, enabling survival of T cells expressing FHVH33-CAR.76
EfficacyUnderstanding antigen loss
Some relapses are either antigen-negative or antigen-low.80 One study in leukemia mouse models could dissectevidence for CAR promoting reversible antigen lossthrough a mechanism called trogocytosis.81 This mecha-nism defines an active process of rapid intercellular trans-fer of membrane fragments and related molecules. Thespecific target antigen is transferred to T cells resulting indecreased density on tumor cells, leading to declined T-cell activity by boosting fratricide T-cell killing andexhaustion.81 These cascades affected CAR constructs thatincluded different costimulatory domains (CD28 or 4-1BB), and the effect was dependent on antigen density.Thus, it was hypothesized that multi-target CAR-T couldovercome these limitations.81
Multi-targetingT-cells expressing single-chain bispecific CAR are able
to prevent antigen escape.68,82 Moreover, CAR pools com-bining two single-input CAR-T products have been pro-posed (Figure 2). Pooling a humanized anti-CD19 and a
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Table 4. Limitations and ways to improve CAR-T therapy in multiple myeloma. What may limit CAR-T therapy? How to improve CAR-T therapy?
Toxicity On-target, on-tumor Anti-IL6 treatment and prevention Safeguard designs incorporating drugs such as rituximab/cetuximab On-target, off-tumor Tackling immunogenicity Simplified CAR structures (e.g., heavy-chain-only binding domains)Resistance Impaired CAR-T expansion/persistence Multi-targting therapy (dual-target, OR-target, CARpool) More accurate measurement of expansion/persistence “Suicide switches” Immunosuppression induced by BM microenvironment Combination of immunomodulatory modulation and CAR-T Senolytic CAR-T (?) Antigen loss or downregulation Address trogocytosis Increase antigen density (e.g. #-secretase-inhibition for anti-BCMA therapy)Management Suboptimal recognition and treatment of severe events Increase comparability and knowledge sharing of intensive care unit management and other care settings Outcome predictionAvailability Lack of scale-up Allogeneic CAR-T High costs Optimize supply chain models (e.g., intermediate players No stockpiling for cryopreservation) TimeBCMA: B-cell maturation antigen; IL6: interleukin-6; BM: bone marrow.
murine anti-BCMA CAR-T was investigated in 22patients.63 The study had a median follow-up of 6 monthsand reported a high ORR of 95%, with CR of 57%, andrelatively low CRS of grades $3 (4%).63 Preliminary resultsof two other CD19/BCMA studies showed similar ORRbut lower CR (22% and 16%).83,84 One study investigateddual-target CAR-T co-expressing two full-length recep-tors, namely CD38 and BCMA.85 Median follow-up was 9months and the ORR was 88%. PFS was 75% and higherCRS of grades $3 were noted compared with tandemCAR (25%). OR-gate tandem CAR consist of a single CARstructure targeting two antigens with two distinct antigenrecognition domains (scFv) linked consecutively with asingle signal transducing intracellular domain.82-86 A recentstudy using CS1/BCMA tandem CAR-T showed superiorCAR expression and function in comparison with T cellsco-expressing individual CS1 and BCMA CAR. Whencompared to the OR-gate (tandem) CAR, dual-target CARrequire a much larger genetic payload, leading to poorertransduction efficiency and reduced proliferation. A recentChinese study using BCMA-CD19 dual FasT CAR-Tshowed an overall response rate of 93.8% with medianduration of follow up of 7.3 months at cutoff. Importantly,most patients showed high-risk features.87 A much morecompact genetic footprint may greatly support viral inte-gration, thus product manufacturing, suggesting an advan-tage for single-chain tandem CAR in relation to dual-tar-geting. With respect to CARpools, this strategy couldavoid poor transduction efficiency. Among these threeapproaches, mechanistically, CAR pool may be the leasteffective.80
Targeting the tumor microenvironmentThe BM milieu is heavily involved in MM pathogenesis
and resistance to treatment. Conflicting data exist onwhether monoclonal antibodies against CD38 are effec-tive in the BM microenvironment,88 whereasimmunomodulatory agents may be able to overcomethese inhibitory effects.89 Accordingly, combining thesedrugs with CAR-T therapy may provide synergisticeffects.59 Conversely, tissue microenvironment itself is
modulated by secretory programs and stable cell-cyclearrest, defined as cellular senescence, which is a tumor-suppressive mechanism. Accumulating aberrant senescentcells create an inflammatory milieu resulting in tissuedamage and fibrosis. In order to eliminate these senescentcells, “senolytic” CAR-T have been proposed.90 One studydiscovered the cell-surface protein urokinase-type plas-minogen activator receptor (uPAR) being broadly inducedduring senescence,88 and further dissected that anti-uPARCAR-T efficiently ablated senescent cells in vitro and invivo, restoring tissue homeostasis in mice with liver fibro-sis.90 In MM, it has been shown that u-PAR contributes tothe functioning of cancer-associated fibroblasts duringMM progression,91 and that higher expression of u-PARwas associated with disease progression, worse survivaland early extramedullary spread of MM cells. Although ithas to be noted that a caveat of senolytic CAR-T are thepotential off-target toxicities,92 these results may encour-age the incorporation of cellular strategies specificallyaddressing the MM microenvironment.
Availability and managementAllogeneic chimeric antigen receptor T cells
Allogeneic CAR-T may decrease cost and enable broad-er availability.93 Notwithstanding, allogeneic CAR-T barethe risk for graft-versus-host disease (GvHD). For this rea-son, TALEN- and CRISPR-based gene editing has beenintroduced to produce allogeneic CAR-T with off-the-shelf availability.68,93,94 One recent study on allogeneic anti-BCMA CAR-T used gene editing, namely TALEN, to con-fer resistance to lymphodepletion and to reduce GvHDrisk.95 By further incorporating a CD20 mimotope-basedswitch-off within the CAR, rituximab could be given toeliminate the CAR-T in case of adverse events. Anotherpreclinical approach using similar safety features but anti-CS1 CAR-T (UCARTCS1),35 specifically degranulated andproliferated in response to MM cells, supporting furtherevaluation and testing of this universal therapy. Currentinvestigational studies also include (i) the non-viralpiggyBac system, aimed at transposing stem cell memoryT cells together with (ii) the Cas-CLOVERTM gene editing
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Figure 2. Overview of current multi-targeted chimeric antigen receptor T-cell approaches. Multi-targeting may be a mean to improve efficacy of CAR-T. Three majormethods could be exploited: OR-gate (tandem) CAR-T: consist of the expression of two different CAR on the same T cell; dual-target CAR-T: consist of encoding twodifferent target specific single-chain variable fragment antibodies on same CAR protein using a single vector; CARpool: involves production of two separate single tar-get CAR-T products infused together or sequentially.
system consisting of CRISPR guide-RNA and dead Cas9fused to Clo51 nuclease, and (iii) a nano-particle deliverysystem carrying the gene for an anti-BCMA Centyrin-based CAR with a fully human binding domain.Rimiducid, a lipid-permeable tacrolimus analogue andprotein dimerizer, may be administered for safety switchactivation.96 Two phase I dose escalation and cohortexpansion studies have recently been initiated. TheCTX120 (clinicaltrials gov. Identifier: NCT04244656) isusing anti-BCMA allogeneic CRISPR-Cas9-engineered Tcells and the PBCAR269A (clinicaltrials gov. Identifier:NCT04171843) which applies its own gene editing tool(ARCUS) for GvHD risk reduction.
Integration of chimeric antigen receptor T cellsinto clinical routine – FACT-JACIE* standardsand EBMT** guidelines
Since 2018, with version 7.0, the *Joint AccreditationCommittee of ISCT and the **European Bone MarrowTransplantation Group (EBMT) (JACIE) prerequisites forcell therapy accreditation have included standards forinfusions of immune-effector cells and CAR-T. The cur-rent recommendation is that CAR-T should be adminis-tered within the framework of an accredited allogeneictransplant program. The Foundation for the Accreditationof Cellular Therapy (FACT)-JACIE do not cover the man-ufacturing of CAR-T but do include supply chain and han-dover of responsibilities when the product is provided bythird party. Overall, JACIE standards are structured on thebasis of three major functional areas in cellular therapy:cell collection, laboratory processing, and clinical program.All areas required dedicated and highly qualified person-nel. Accredited programs for cell therapy must implementa product labeling system that guarantees identificationand traceability from collection to manufacturing site andreturn to clinical units. EBMT recommendations furtherstress the importance of staff training97 and of multidisci-plinary approaches with teams who include transplantphysicians along with qualified internal medicine sub-spe-cialists after a specific education program. Importantly,CAR-T infusions should be coordinated with intensivecare specialists. All accredited centers must implement apolicy for rapid escalation of care for critically ill patientsincluding availability of specific drugs (i.e., tocilizumab).Though complications may vary among CAR-T products,they tend to follow a predictable timeline contributing tobed-planning decisions. Recent reports allow designingprotocols for anti-infective prophylaxis and common post-infusion complications such as infections and tumor lysissyndrome.98 Inevitably, the unfortunate COVID-19 pan-demic stresses the importance of scrupulous adherence to
recommended hygiene procedures.99 Importantly, anEBMT registry, for all transplant centers accredited for celltherapies, has been created to collect date on efficacy, sideeffects and clinical outcomes for post-marketing surveil-lance.
Conclusions
The clinical role of CAR-T in the current armamentari-um of MM treatments remains as yet to be fully deter-mined. Moreover, other promising forms of antibody-based immunotherapies have been added. Despite somelimitations of CAR-T therapy experienced in early studiesin MM, one advantage of this cellular therapy is the inher-ent potential to finetune its design. Simpler structures andmulti-target approaches may significantly improve effica-cy and safety. Constant learning to handle CAR-T therapymay also enable better patient-centered management.Last, long-term outcome studies and specific detectionand analysis of CAR-T dynamics in vivo are essential toallow deeper understanding of their inherent functionswhich will facilitate future designs of improved CAR-Tproducts. However, selecting patients who may most ben-efit from CAR-T and best timing of their administrationstill require rather lengthy and thorough clinical investiga-tions. One more challenge that lies ahead will be the costeffectiveness of future commercial products. This issuehas already been addressed in patients with lymphomawhere cost reductions will be inevitable to make CAR-Tsustainable therapies for health care systems.100 Despite allremaining open questions and issues that still need to beaddressed, and hopefully answered and resolved withinthe next years, we are now, without any doubt, at thedawn of a new era that will significantly improve patientoutcome.
DisclosuresNo conflicts of interest to disclose.
ContributionsBB, ME, NWCJD designed the review and wrote the manu-
script; LG, MD, FG, ET, LGRL, SD, NG, NK, RP and ETprovided data and interpretation; MAD, PS, HE and MBreviewed the manuscript.
FundingLGRL as BITRECS fellow has received funding from the
European Union’s Horizon 2020 research and innovation pro-gramme under the Marie Sklodowska-Curie grant agreementNo 754550 and from “La Caixa” Foundation. SD has receivedfunding from the Mildred Scheel Early Career Center funded bythe German Cancer Aid.
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CAR T cell therapies in multiple myeloma
haematologica | 2021; 106(8) 2065
Vol.:(0123456789)1 3
Cancer Immunology, Immunotherapy (2019) 68:365–377 https://doi.org/10.1007/s00262-018-2281-2
O R I G I N A L A R T I C L E
CRISPR/Cas9-mediated PD-1 disruption enhances human mesothelin-targeted CAR T cell e!ector functions
Wanghong!Hu1!· Zhenguo!Zi1!· Yanling!Jin1!· Gaoxin!Li1!· Kang!Shao2!· Qiliang!Cai3!· Xiaojing!Ma1!· Fang!Wei1
Received: 24 January 2018 / Accepted: 1 December 2018 / Published online: 6 December 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018
AbstractThe interaction between programmed cell death protein 1 (PD-1) on activated T cells and its ligands on a target tumour may limit the capacity of chimeric antigen receptor (CAR) T cells to eradicate solid tumours. PD-1 blockade could potentially enhance CAR T cell function. Here, we show that mesothelin is overexpressed in human triple-negative breast cancer cells and can be targeted by CAR T cells. To overcome the suppressive e!ect of PD-1 on CAR T cells, we utilized CRISPR/Cas9 ribonucleoprotein-mediated editing to disrupt the programmed cell death-1 (PD-1) gene locus in human primary T cells, resulting in a significantly reduced PD-1hi population. This reduction had little e!ect on CAR T cell proliferation but strongly augmented CAR T cell cytokine production and cytotoxicity towards PD-L1-expressing cancer cells in"vitro. CAR T cells with PD-1 disruption show enhanced tumour control and relapse prevention in"vivo when compared with CAR T cells with or without #PD-1 antibody blockade. Our study demonstrates a potential advantage of integrated immune checkpoint blockade with CAR T cells in controlling solid tumours and provides an alternative CAR T cell strategy for adoptive transfer therapy.
Keywords Mesothelin"· Chimeric antigen receptor"· Programmed cell death protein 1"· SgRNA-guided clustered regularly interspaced short palindrome repeats-associated nuclease Cas9
Abbreviations4-1BB TNF Receptor superfamily member 9CRISPR/Cas9 sgRNA-guided clustered regularly inter-
spaced short palindrome repeats-associ-ated nuclease Cas9
Meso MesothelinRFP Red fluorescence protein
RNP RibonucleoproteinT7E1 T7 endonuclease ITim-3 T cell immunoglobulin and
mucin-domain-containing-3TNBC Triple-negative breast cancer
Introduction
The chimeric antigen receptor (CAR) is an artificial mol-ecule containing an antibody-based extracellular structure and cytosolic domains that encode signal transduction mod-ules of the T cell receptor [1]; the CAR targets T cells in an MHC-unrestricted manner, regardless of the status of co-stimulatory factors. The impressive clinical outcomes asso-ciated with the adoptive transfer of CAR-expressing T (CAR T) cells in B-lineage malignancy therapy has prompted its application in the treatment of solid tumours. However, e!orts to apply the CD19-targeting CAR therapy for haemat-opoietic malignancies towards the treatment of solid tumours has encountered di$culties [2]. The development of a suit-able T cell population that appropriately recognizes cancer antigens without causing detrimental e!ects to normal cells remains a major challenge [3].
Wanghong Hu, Zhenguo Zi and Yanling Jin contributed equally.
Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0026 2-018-2281-2) contains supplementary material, which is available to authorized users.
Fang Wei [email protected] Sheng Yushou Center of"Cell Biology and"Immunology,
School of"Life Sciences and"Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Minghang, Shanghai"200240, China
2 A$liated Renji Hospital, Shanghai Jiao Tong University, Shanghai, China
3 MOE and"MOH Key Laboratory of"Medical Molecular Virology, School of"Basic Medicine, Shanghai Medical College, Fudan University, Shanghai, China
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Mesothelin (Meso) is a glycosylphosphatidylinositol-anchored cell surface protein that is expressed at low levels on normal mesothelial cells. Meso knockout mice exhibit normal development and reproduction [4]. Clinical and preclinical data have shown that Meso is overexpressed in mesothelioma, ovarian cancer, pancreatic cancer, and other cancers. Its aberrant expression not only promotes cancer cell proliferation but also contributes to tumour invasion, metastasis and drug resistance; therefore, Meso has emerged as an attractive target for cancer immunotherapy [5]. Several Meso-targeted immunotherapy clinical trials have shown the safety of this treatment [6]. Unlike breast cancer that expresses steroid receptors or HER-2/neu cancer, which responds to many treatments, triple-negative breast cancer (TNBC, which is oestrogen receptor-, progesterone recep-tor- and human epidermal growth factor receptor 2-negative breast cancer) is largely unresponsive to the clinically availa-ble targeted therapies and frequently has the worst prognosis among breast tumour subtypes [7]. Meso is overexpressed in 34–67% of TNBC patients [8–10] but is relatively rare in ER+ or Her2/neu+ breast cancer patients. Meso-positive TNBC patients develop more distant metastases and have a shorter disease-free interval with significantly lower overall and disease-free survival rates than Meso-negative TNBC patients [11]. Tchou et"al. have previously shown that Meso CAR T cells display cytotoxic activity towards TNBC cell lines highly expressing Meso [9], but their e!ector function requires further characterization.
It is well accepted that tumours adapt immune regula-tory signalling pathways to evade immune recognition and elimination [12]. One of the mechanisms utilized by tumours is the upregulation of programmed death-ligand 1 (PD-L1), which has been identified as an indicator of poor progno-sis in several tumour types, including breast cancers. By engaging programmed death 1 (PD-1) receptors on activated T cells, PD-L1+ tumours direct tumour-specific T cells, including adoptively transferred T cells, towards functional exhaustion. We have shown that PD-1 expression causes a step-wise loss of proliferative capacity, cytokine secretion, and finally the cytotoxic activity of T cells [13]. Even with a co-activation signal from 4-1BB, tumour-infiltrated Meso CAR T cells still show a hypofunctional phenotype and rap-idly lose their cytokine secretion and cytotoxic abilities. This loss of function is associated with increased expression of the surface inhibitory receptor PD-1, which has been con-firmed by PD-1 blockade in"vitro with a PD-L1 neutralizing antibody [14]. We hypothesized that blockade of the e!ector function of CAR T cells with PD-1 could be enhanced. Anti-PD-L1 and PD-1 antibodies have been used in clinical trials and in the treatment of di!erent cancer types. Despite the success of monoclonal antibody blockade of PD-1 in human cancer treatment, most patients do not have favourable clini-cal responses [15, 16]. In"vivo imaging data have revealed
that this result is partially because anti-PD-1 monoclonal antibodies are captured prior to reaching the T cell surface by PD-1 tumour-associated macrophages through their Fc domain, removing their ability to block PD-1 and resulting in suppression of T cell function [17]. Several other strate-gies have also been applied for PD-1 blockade, including PD1/CD28 converters [18, 19], PD-1 dominant-negative receptors [20] and PD-1 knockout by the CRISPR/Cas9 sys-tem [21]. The benefit of combining PD-1 blockade treatment with CAR T cells has been observed in several reports [19]. Administration of PD-1 blocking antibody to patients with refractory di!use large B cell lymphoma and progressive lymphoma has been e!ective in preventing the failure of CD19-directed CAR T cell therapy [22]. The combination of PD-1 disruption through CRISPR/Cas9 and CD19-CAR T cell therapy against B cell lymphoma and other models has been studied [23–25], but whether PD-1 disruption would improve CAR T cell therapy in a breast cancer model has not been fully reported in the literature.
With the goal of overcoming the tumour PD-L1 e!ects on adoptively transferred T cells, we sought to evaluate T cell e!ector functions by PD-1 knockdown mediated by a CRISPR/Cas9 approach. Here, we show that CAR T cells that specifically recognize Meso can target Meso-expressing tumours (TNBC) and that this e!ect is further enhanced by combination with CRISPR/Cas9-mediated PD-1 genome modification.
Materials and!methods
T cell isolation, gene transfer, and!expansion
Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll-Paque PLUS (GE Healthcare) density centrifuga-tion. The CD4 or CD8 T cells were isolated and expanded as previously described by Wei et"al. [13].
Plasmids
Sequences of the Meso-specific target scFv with a CD8 lead-ing sequence, a CD8 hinge and transmembrane sequence, and 4-1BB and CD3% signalling domains were synthesized (Thermo Fisher Scientific) and inserted into a lentiviral vec-tor. A DNA fragment containing a PD-1 genomic exon 1 targeting sequence was synthesized, ligated with a manu-facturer-provided sgRNA oligonucleotide template and then inserted into the pMDTM 19"T-vector.
Antibody staining and!flow cytometry
Surface and intracellular cytokine staining was performed as described above. Data were obtained with an Accuri™
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C6 flow cytometer (BD Biosciences) and analysed with FlowJo software (Tree Star Inc.). The following antibodies were used for cell surface and intracellular staining: APC-PD1 (eBioscience, clone J105), FITC-Meso (R&D, clone 420411), FITC-CD4 (BD Biosciences, clone RPA-T4), PE-CD25 (BD Biosciences, clone 4E3), PerCP-CD8 (BD Biosciences, clone RPA-T8), PE-IL-2 (BD Biosciences, clone MQ1-17H12), APC-IFN& (BD Biosciences, clone B27), CD3 (BD Biosciences, clone UCH71), and PE-CD107a (BD Biosciences, clone H4A3) antibodies. To detect CAR expression, cells were stained using goat anti-mouse IgG-biotin (Jackson ImmunoResearch) followed by streptavidin-PE (BD Biosciences).
Cytotoxicity assays
The cytotoxic capacity of CAR T cells was tested in an 18-h luciferase-based killing assay as described elsewhere [26]. Lysis e$ciency was calculated as [1 ' (RLUsample)/(RLUmax)] ( 100.
PD-1 sgRNA-Cas9 RNP assembly and!electroporation
PD-1 sgRNA was transcribed in"vitro under the control of a T7 promoter, further purified, and then assembled with GeneArt™ Platinum™ Cas9 Nuclease (Thermo Fisher Scientific). At day 5 after activation, PD-1 sgRNA was electroporated into T cells with a Neon transfection kit and device (Thermo Fisher Scientific).
Analysis of!genome editing and!PD-1 gene expression
Editing e$ciency was estimated by PD-1 surface stain-ing and a T7 endonuclease I assay. At day 14, T cells were stimulated by Dynabeads® Human T-Activator CD3/CD28 (Thermo Fisher Scientific) and cultured in a 37"°C incuba-tor for 48"h. Then, PD-1 and CD25 staining was performed. Data were analysed by flow cytometry. A T7E1 assay was performed following the manufacturer’s instructions. The CRISPR/Cas9 editing e$ciency was calculated by the fol-lowing equation: % gene modification = 100 ( (1 ' (1 ' frac-tion cleaved)1/2).
Xenograft assays
A total of 2 ( 106 firefly luciferase-expressing BT549 cells were injected into the fourth mammary gland of NOD-Prk-dcscid IL2rgnull (NSG) mice. Mice were monitored weekly for tumour growth by bioluminescence imaging with a Xenogen Spectrum system and Living Image software ver-sion 3.2 (Calipere PerkinElmer, Hopkinton, MA, USA) fol-lowing a previously described protocol [27]. At day 25, T
cells were administered at a dose of 1 ( 105 Meso CAR + T cells/mouse through the tail vein. PD-1-blocking antibody (clone EH12, Biolegend) was injected at 2"mg/kg every 2 weeks through the tail vein. Peripheral blood was drawn from the tail vein and stained with anti-human CD45 anti-body (clone H130, Biolegend) at the indicated experimental time points and mixed with CountBright™ Absolute Count-ing Beads (Thermo Fisher Scientific) as an event collecting control. The samples were analysed by flow cytometry, and cells were quantified by the number of hCD45+ cell events/the number of bead events ( number of beads per test/test volume.
Results
Mesothelin and!PD-L1 are co-expressed in!BT-549 and!HeLa cell lines
To study whether gene editing at the pdcd-1 locus would influence the expression of PD-1 and enhance CAR function in T cells, we evaluated Meso and PD-L1 expression levels in MCF7 and three available TNBC lines (MDA-MB-231, BT-549 and Hs 578T). Using HeLa cells as a reference (cell line from which Meso as an antigen was cloned and with which monoclonal antibodies are screened to construct the CAR molecule) [28, 29], Meso was detected in BT-549 cells but not in MCF7, MDA-MB-231 and Hs 578T cells by both Western blot (Fig."1a) and flow cytometry (Fig."1b). This finding is consistent with that of previous reports, suggest-ing that Meso is not expressed in MDA-MB-231 cells [9]. The flow cytometry data (Fig."1b) showed that all cell lines expressed PD-L1 at various levels. Therefore, we chose Meso+PD-L1+ BT-549 and HeLa cells as putative target cells and Meso'PD-L1+ MCF7 cells as non-target cells to evaluate Meso-specific CAR T cell function.
Characterization of!the!e"ector function of!Meso CAR T cells
Carpenito et"al. constructed a Meso-targeted CAR contain-ing di!erent signalling domains [30]. CAR T cells with a 4-1BB signalling domain showed the greatest persistence in a tumour model [31] and advanced toward towards a central memory phenotype [32]. We generated a Meso CAR mol-ecule with a Meso-binding scFv domain, 4-1BB intracellular and TCR-% signal transduction domains and other required regions in a lentiviral vector (Supplementary Figure"1a) and confirmed Meso CAR expression in human primary T cells (Supplementary Figure"1b).
To investigate the antitumour e!ects of the Meso CAR T cells, cytotoxicity was measured in both an indirect way by surface CD107a staining (Supplementary Figure"2a) [33]
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and a direct way by a luciferase-based cytotoxic T lym-phocyte (CTL) assay [26]. We transduced MCF7, BT-549 and HeLa cells with a lentivirus containing DNA encod-ing both red fluorescent protein (RFP) and luciferase. Cells were sorted based on RFP expression, and a cell line stably expressing luciferase was generated as a target for luciferase-based CTL assays (Fig."2a). At day 9 after stimu-lation, Meso CAR T cells were mixed with target cells at various e!ector:target (E:T) ratios and co-incubated for 18"h (Fig."2b). We observed that 40% of BT-549 cells and 58% of HeLa cells were lysed even at a low E:T ratio of 0.5:1 and that the cytotoxic activity of CAR T cells increased with increasing E:T ratios.
The antitumour e!ects of CAR-redirected T cells depend on their capacity to secrete cytokines after exposure to anti-gens. Therefore, cytokine secretion from Meso CAR T cells was measured by intracellular cytokine staining (Supple-mentary Figure" 2b) and enzyme-linked immunosorbent assay (ELISA) after exposure to target cells. We detected
robust IFN& (Fig." 2c) and IL-2 (Fig." 2d) secretion when CAR T cells were co-incubated with BT549 or HeLa cells but not with MCF7 cells.
These results show that Meso CAR T cells had potent e!ects on Meso-expressing TNBC cells.
Ablation of!the!T cell negative regulator PD-1 with!Cas9 ribonucleoproteins (RNPs)
Expression of the exhaustion marker/inhibitory recep-tor PD-1 has been observed in 19BBz-CAR T cells, even though 4-1BB pathways showed a reduced induction of PD-1 expression [31]. Due to the upregulation of PD-1 expression on the surface, tumour-infiltrated CAR T cells could rapidly lose their antitumour cell function [14]. To simultaneously express CAR and knockout pdcd1, we first stimulated and transduced human T cells (CD4 mixed with CD8 at a ratio of 1:1) as described previously [13] and subsequently electroporated PD-1 sgRNA/Cas9 RNP
Fig. 1 Evaluation of mesothelin and PD-L1 expression in various cancer cell lines. a Mesothelin expression levels were evaluated by Western blot. b Histograms depicting mesothelin and PD-L1 expres-sion (red line) in the breast cancer oestrogen and progesterone recep-
tor-positive line MCF7 and the TNBC lines MDA-MB-231, BT-549 and Hs 578" T. HeLa cells served as a positive control for mesothe-lin. Isotype staining served as a negative control for mesothelin and PD-L1 staining (solid grey with dotted line)
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assembly 5 days later when the pdcd1 locus was still open for PD-1 expression (a protocol for combined lentiviral and Cas9 RNP transduction of human primary T cells is schematically shown in Fig."3a, and the targeting locus is indicated in Fig."3b). IL-7 (5"ng/ml) and low concentra-tions of IL-2 (10"U/ml) were added to the culture medium to yield a better cell survival rate after electroporation. The T cells were re-stimulated by Dynabeads® Human T-Activator CD3/CD28 at day 12 and stained with anti-PD-1 antibody 2 days later. PD-1 expression was analysed within the activated cell population (gated with CD25+). A 59.2 ± 9.0% (n = 6) reduction in the PD-1hi population was observed in Meso CAR/PD-1 sgRNA-Cas9 RNP
cells when compared with Meso CAR T cells (Fig."3c). A 30.8 ± 5.0% (n = 5) disruption was also confirmed with the T7E1 assay (which might underestimate knockout e$-ciency) (Fig."3d) [21]. There was no di!erence in PD-1 disruption e$ciency between the Meso CAR + and Meso CAR ' populations (Supplementary Figure"3). To deter-mine whether PD-1 disruption would impact T cell per-sistence, we focused on the fraction of CD8 T cells after in"vitro expansion (Fig."3e). A bias towards CD8 T cells was noted in control cells, since IL-2 was added to the cul-ture medium to promote CD8 T cell growth. Interestingly, the addition of IL-2 did not increase the fraction of CD8 T cells in Meso CAR-expressing T cells as significantly
Fig. 2 Meso CAR T cell e!ects on mesothelin-expressing tumour cells. a Luciferase-RFP stably transduced cell lines (MCF7, BT-549, and HeLa) were generated (red line). The untransduced parent cell lines are depicted with black lines. Lower panel: b flow cytometry-based cytotoxicity assay. Control or Meso CAR T cells were co-incu-bated with di!erent targets for 18" h at the indicated E:T ratios. Pri-mary human T cells were transduced with lentivirus encoding Meso
CAR. Nine days later, T cells were co-cultured with the indicated cells. The cytotoxicity of T cells was measured by luciferase assay. c Levels of interferon-& (IFN-&) and d interleukin-2 (IL-2) were meas-ured by enzyme-linked immunosorbent assay (ELISA) 24"h after cul-turing control or Meso CAR T cells with MCF7, BT-549 and HeLa at E:T ratios of 1:1 and 1:2. Data are reported as the mean from three independent experiments. Error bars indicate the SEM
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as in the control cells. Even with the 4-1BB signal, which supports a moderate rise in the CD8 T cell fraction, the Meso CAR T cell groups (with or without PD-1 sgRNA-Cas9 RNP) showed lower CD8 counts than the control group, which suggests that the electroporation procedure might a!ect the growth of CD8 T cells more than that of CD4 T cells. The Meso CAR/PD-1 sgRNA-Cas9 RNP
T cells showed the lowest CD8 fraction, which indicated that PD-1 knockout might a!ect CD8 T cell expansion for unknown reasons. No di!erence was observed in the transduction e$ciency of Meso CAR in individual CD4 or CD8 subsets between Meso CAR/PD-1 sgRNA-Cas9 RNP and Meso CAR only groups (data not shown).
Fig. 3 PD-1 gene editing by sgRNA/Cas9 RNP assembly in primary human T cells. a Schematic representation of the experimental proto-col of introducing PD-1 sgRNA/Cas9 RNP assembly and CAR into T cells. b Schematic representation of the PD-1 sgRNA/Cas9 RNP assembly-targeted site in the human PDCD-1 genome. c PD-1 expres-sion is shown by histograms of PD-1 cell-surface staining. The PD-1 sgRNA/Cas9 RNP assembly was electroporated into T cells 5 days
after stimulation, and PD-1 expression was then evaluated on day 2 after the second stimulation. The mean fluorescence intensity of PD-1 staining is shown above each sample. (*p < 0.05, **p < 0.01, Stu-dent’s t test). d The T7E1 assay was used to confirm successful edit-ing in the PD-1 genome locus. e PD-1 editing significantly a!ects the CD8 T cell fraction (*p < 0.05, **p < 0.01, Student’s t test). CD8 T cells were analysed by flow cytometry
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PD-1 disruption in!CAR T cells enhances their ex!vivo e"ector functions
To determine whether PD-1 disruption influences T cell activity, CAR T cells with/without PD-1 disruption were washed extensively with phosphate-buffered saline to remove the residual IL-2"at day 14 and then co-incubated with MCF7, BT-549 or HeLa cells at a ratio of 1:1 unless indicated otherwise. When BT-549 and HeLa cells were co-cultured with T cells expressing Meso CAR for 18"h, they showed 11.44- and 3.8-fold increases in PD-L1 expression, respectively (Fig."4a). Expression of PD-L1 in tumour cells could be further stimulated (without signifi-cant di!erences) when the tumour cells were co-cultured with Meso CAR/PD-1 sgRNA-Cas9 RNP T cells. No ele-vation in PD-L1 expression was observed in MCF7 cells. We next examined the cytokine production and cytotoxic capacities of Meso CAR/PD-1 sgRNA-Cas9 RNP T cells. When exposed to antigens expressed on BT-549 and HeLa cells at 2 di!erent ratios, Meso CAR/PD-1 sgRNA-Cas9 RNP T cells showed significantly higher antitumour activ-ity (Fig."4b) than Meso CAR T cells; however, both groups showed a reduction in cytotoxicity due to the extended cell culture time. The actual enhancement of cytotoxic capacity per cell could be greater considering the lower CD4/CD8 ratio shown in Fig." 3e. Meso CAR/PD-1 sgRNA-Cas9 RNP T cells showed significantly elevated IFN-& and IL-2 levels (Fig."4c) compared with control CAR T cells. The elevated IFN-& levels were also reflected by the increased expression of PD-L1 in tumour cells, since IFN-& could stimulate PD-L1 expression. We did not observe improved in" vitro expansion from PD-1 disruption when Meso CAR/PD-1 sgRNA-Cas9 RNP T cells were stimulated by BT-549" at day 14 and day 19 (indicated as the 1st and 2nd time points) after the first expansion following the application of Dynabeads® Human T-Activator CD3/CD28 (Fig."4d), which suggested that PD-1 disruption may have di!erential e!ects on di!erent T cell e!ector functions, which may be correlated with our previous finding [13]. There was no obvious alteration in the levels of other co-inhibitory factors, including CTLA-4 and Tim-3 (Fig."4e), which suggested that the enhanced e!ects were attributed to only PD-1 Cas9 RNP-induced PD-1 disruption.
As PD-1 blocking antibodies have demonstrated ben-eficial e!ects in the treatment of various malignancies, including several solid tumours, we next compared the abilities of PD-1 editing by Cas9 RNP and PD-1 blocking antibody (clone EH12.2H7) to rescue CAR T cell function (Fig."5). PD-1 disruption by sgRNA-Cas9 RNP exhibited significantly higher potency in enhancing cytotoxicity and the production of cytokines, IL-2 and IFN& by Meso CAR T cells when compared that by antibody blockade.
PD-1-disrupted CAR T cells exhibit better tumour control in!vivo
Next, we evaluated whether Meso CAR/PD-1 sgRNA-Cas9 RNP T cells would provide an in" vivo advantage in the orthotopic xenograft NOD-Prkdcscid IL2rgnull (NSG) mouse model utilizing BT-549 cells stably expressing luciferase. The treatment response was monitored by tumour burden measurements (bioluminescence imaging, BLI) and T cell persistence (hCD45+ cell counts and CAR genomic copies). A low dose (1 ( 105 per mouse) of Meso CAR + T cells was injected into mice with a pre-established tumour burden. Meso CAR/PD-1 sgRNA-Cas9 RNP T cells showed a better antitumour e!ect than Meso CAR T cells (Fig."6a), with a significant decrease in tumour BLI at day 18 (Fig."6b). An increasing hCD45+ cell count was observed during the clear-ance of tumour cells, but the di!erence was not significant between groups (Fig."6c); however, there was a significant increase in the number of Meso CAR genomic copies at day 18 (Fig."6d), which suggested better expansion of Meso CAR T cells in" vivo with PD-1 editing. To compare the e!ect of the PD-1 blocking antibody vs. that of PD-1 editing on rescuing CAR T cells, EH12 antibody was subsequently administered intraperitoneally at a concentration of 2"mg/kg to the indicated group starting at day 1 every 2 weeks fol-lowing the clinical protocol described previously [22]. The results showed that PD-1 blocking antibody did not improve the e!ect of CAR T cells in our model (Fig."6a, b). Tumour relapse was observed in all groups at day 44 and was the most severe in the Meso CAR/#PD-1 group and the least severe in the Meso CAR/PD-1 sgRNA-Cas9 RNP group. To clarify whether the relapse was caused by antigen loss in the tumour cells or a lack of T cell persistence, we sacrificed mice and dissected the tumour (Fig."6e) to analyse hCD3+ T cell infiltration (Fig."6f) and the expression of Meso in the tumour (Fig."6g). We observed significantly greater hCD3+ T cell infiltration in the tumour of Meso CAR/PD-1 sgRNA-Cas9 RNP T cell group than in that of the Meso CAR T cell group. A decrease in Meso expression was observed in the majority of samples, which indicated that the relapse might have resulted from the loss of antigen. However, we also observed increased expression of Meso in two out of four samples in the Meso CAR T cell/#PD-1 group, along with the presence of CD3 T cells in the tumour and Meso CAR copies in the peripheral blood. These observations suggest that T cell exhaustion may have occurred in the Meso CAR T cell/#PD-1 group. By the end of the experiment, we did not observe any autoimmune symptoms among all treated groups.
Thus, PD-1 disruption through CRISPR Cas9 enhanced the capacity of CAR T cells to control large established tumours.
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Discussion
Although two major cancer immunotherapy approaches, CAR-engineered T cells and blockade of co-inhibitory fac-tors, have provided cancer patients with more hope for cura-tive therapies, control of disease progression in many cancer types, especially solid tumours, remains a great challenge. CAR T cells have been reported as hypofunctional in the tumour microenvironment [14] partially due to the suppres-sive e!ect of their own expression of PD-1. The combination of CAR T cells and PD-1 blockade could improve tumour control and overall survival [34]. PD-1 blockade achieved by blocking antibodies, dominant-negative receptors, or chi-meric PD-1 switching molecules may improve CAR T cell e$cacy. The blockade e$cacy of PD-1 blocking antibody is short-lived and relies upon repeated administration [15, 20].
Recently, reports have shown that CRISPR/Cas9-mediated disruption of PD-1 could boost the function of CD19-CAR T cells [24, 35] and that Epstein–Barr virus LMP2 peptide increased the cytotoxic function of T lymphocytes to di!er-ent degrees [25]. In the present study, we chose to introduce PD-1 sgRNA/Cas9 RNP cells after a single stimulation with #CD3/CD28 beads, which resulted in shorter in"vitro culture time and preservation of Cas9 editing e$ciency when com-pared with two longer stimulations with #CD3/CD28 beads using the protocol provided by Rupp et"al. [24]. We also showed that Meso CAR T cells could be genetically edited at the PD-1 locus, enhancing their capacities for cytotoxic-ity and cytokine secretion against TNBC. In particular, we observed a 2- to threefold increase in cytotoxicity to BT549 cells, which is a much greater enhancement compared with that in a previous report [31]. However, this gene editing protocol did not alter the proliferative ability of the cells or the expression of the co-inhibitory factors CTLA-4 and Tim-3.
It has been shown that di!erent Meso antibody clones can display variable reactivity on the cell membrane and in the cytoplasm of neoplastic cells in the same samples [36]. The inconsistency between the poor surface protein staining by flow cytometry and strong Meso protein staining by Western blot suggests that our antibody may not e$ciently bind the cell surface or that there is less surface expression of Meso. Nevertheless, a dramatic di!erence in the responses of Meso CAR T cells to di!erent Meso expression levels in target cells was clearly observed, both in terms of cytotoxicity and cytokine production. Interestingly, June’s group observed that CTL019 (CD19-CAR T) cells were cytotoxic to mul-tiple myeloma neoplastic cells and could be successfully utilized in the treatment of one patient with refractory mul-tiple myeloma [37]; however, the patient’s neoplastic plasma
Fig. 4 Enhanced Meso CAR T cell e!ect upon PD-1 gene disrup-tion. a Increased expression of PD-L1 in the cancer cell lines MCF7, BT-549 and HeLa was observed when cells were co-cultured sepa-rately with Meso CAR or Meso CAR/PD-1 sgRNA/Cas9 RNP T cells for 18"h. (*p < 0.05, **p < 0.01, Student’s t test). b The cytotox-icity of T cells was measured by luciferase assay. c After co-culture for an additional 30" h, the supernatant was collected, and the ability of these stimulated T cells to produce IFN& and IL-2 was measured by enzyme-linked immunosorbent assay. b, c Data (mean ± SEM) are representative of at least 3 independent experiments. Statisti-cally significant e!ector function enhancement in PD-1-edited CAR T cells was observed at 2 di!erent E:T ratios (*p < 0.05, **p < 0.01, Student’s t test). d At day 14, T cells were stimulated by BT-549 cells (first stimulation). Five days later, the cells were re-stimulated by BT-549 cells (second stimulation). The ratio of BT-549:T cells was 1:3, and surviving T cells were counted after trypan blue stain-ing. Data are representative of at least three independent experiments with di!erent donors and plotted as the mean ± SEM. e CTLA-4 and Tim-3 expression in T cells was compared between Meso CAR (dark line) and Meso CAR/PD-1 sgRNA/Cas9 RNP T cells (red lines)
!
Fig. 5 PD-1-edited CAR T cells show better cytotoxicity and cytokine production than PD-1 blocking antibody-treated CAR T cells. Meso CAR T cells with and without PD-1 sgRNA/Cas9 RNP
or #PD-1 blocking antibody treatment were used in functional assays. The #PD-1 blocking antibody EH12.2H7 was added at 10" µg/ml when T cells were mixed with BT549 cells
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Fig. 6 PD-1 editing shows better synergy with the CAR in T cell-mediated tumour control in"vivo when compared with PD-1 blocking antibody treatment. a Enhanced CAR T cell tumour control capac-ity with sgRNA/Cas9 RNP-mediated PD-1 disruption in NSG mice with a BT549 orthotopic xenograft model. b The enhanced e$cacy was measured by BLI in each group of mice. c Human CD45+ cells in peripheral blood collected from the tail vein were separated by flow cytometry and quantified by CountBright™ Absolute Count-ing Beads. The results are expressed as the mean count ± SEM with
n = 8 for each group. d Meso CAR copies were measured in genomic DNA extracted from mouse blood samples at the indicated time points by absolute quantitative PCR (*p < 0.05, **p < 0.01, Student’s t test). e Tumours were dissected from mice at day 44 after CAR T cell treatment. f hCD3+ cells were quantified from single-cell suspen-sions from collagenase VIII- and DNase-digested tumour samples (*p < 0.05, **p < 0.01, Student’s t test). g Expression of mesothelin in dissected tumours from each group was measured by Western blot
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cells expressed extremely low levels of CD19. This finding suggests that the e$cacy of immunotherapy treatments in targeting shared antigens, especially in the heterogeneous solid tumour background, may need additional evaluation.
Upon PD-1 disruption, Meso CAR T cells showed increased cytotoxicity and cytokine production but not increased proliferation in"vitro. This observation could be related to the low expression levels of Meso at the surface of the tumour cells. This observation could also be correlated with our previous findings [13]. The capacity of T cell pro-liferation was more suppressed than other T cell functions by PD-1-mediated signals when PD-L1-expressing artifi-cial antigen-presenting cells (K562) were used to stimulate resting SL9 TCR-expressing T cells with barely detectable levels of endogenous PD-1 [13]. We observed no alterations in the expression of the co-inhibitory factors CTLA-4 and Tim-3. In a lymphocytic choriomeningitis virus (LCMV) chronic infection and tumour model, exhausted T cells showed remarkably stable exhaustion-associated DNA meth-ylation programming, which restricted T cell clonal expan-sion in response to PD-1 blockade therapy [38]. These data suggested that alterations in the PD-1/PD-L1 axis did not influence the DNA methylation status across the genomic region related to proliferation. Therefore, T cell PD-1 dis-ruption using our approach may not have improved T cell proliferation via changes to DNA methylation status, which needs further elucidation.
The phenomenon of relapse after CAR T cell treatment has been observed in various clinical reports. Our data sug-gested that PD-1 disruption through CRISPR/Cas9 could not only enhance tumour control by Meso CAR T cells but also delay tumour relapse to some degree after CAR T cell treatment. Our study provides evidence that the combina-tion of CAR T cells and co-inhibitory factor gene editing is e!ective.
We showed that PD-1 disruption mediated by CRISPR/Cas9 improved the e!ect of the CAR, which contains 4-1BB and CD3 (BBz) as signalling domains, on T cell function. Although Meso 28z and BBz-CAR T cells exhib-ited equivalent secretion of e!ector cytokines and pro-liferation in"vitro upon initial antigen stimulation, Meso 28z-CAR T cells expressed higher levels of exhaustion markers, such as PD-1, TIM-3, and LAG-3, upon further stimulation than Meso BBz-CAR T cells [20]. We antici-pate that PD-1 disruption could further enhance the func-tion of CAR T cells containing the CD28 and CD3 signal-ling domain (28z).
Overall, we have demonstrated that PD-1 disruption mediated by Cas9/CRISPR could improve CAR T cell e!ector function in"vitro and in"vivo. A combination of positive stimulation from the CAR and blockade of nega-tive regulators, such as PD-1, could enhance T cell antitu-mour e!ects against TNBC.
Acknowledgements We are grateful to the blood donors for their con-tribution; Zhouluo Ou for providing the breast cancer cell lines used in this study; Ilya Vinnikov for proofreading the manuscript; and Wei Zhang and Huan Wang for their helpful suggestions.
Author contributions All authors made substantial contributions to the conception and design of this work. WH, ZZ and YJ performed the experiments, analysed the data and wrote the manuscript. KS contrib-uted to blood donor communication and sample collection. GL con-tributed to the molecular biology experiments. QC and XM proofread the manuscript and gave intellectual suggestions. FW contributed to the conception and design, data analysis and interpretation, and manu-script writing and provided financial support. All authors viewed and approved the final version of the manuscript.
Funding This work was supported by the National Natural Science Foundation of China (81402542) and the scholarship of Pujiang Talents in Shanghai to Fang Wei (14PJ1405600).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of interest.
Ethical approval For the use of blood samples from healthy donors, written informed consent was obtained in accordance with the of the Shanghai Jiao Tong Human Sample Committee on March 1, 2014. The mouse study was carried out in accordance with the recommendations of East China Normal University Animal Care guidelines from the East China Normal University Animal Care Committee. All experimental protocols were approved on August 1, 2016, by the East China Normal University Animal Care Committee.
Animal source Six-week-old female NOD-Prkdcscid IL2rgnull (NSG) mice were purchased from Vitalstar, China, and housed in ventilated cages in our pathogen-free facility.
Cell line authentication The human cell lines MCF7, MDA-MB-231, BT-549, Hs 578T and HeLa were kindly provided by Zouluo Ou (Fudan University, Shanghai, China). The cell lines were authenticated by the Chinese Academy of Sciences Committee Type Culture Collec-tion Cell Bank.
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Oncotarget12226www.impactjournals.com/oncotarget
www.impactjournals.com/oncotarget/ Oncotarget, 2018, Vol. 9, (No. 15), pp: 12226-12239
MUC16 overexpression induced by gene mutations promotes lung cancer cell growth and invasion
Madiha Kanwal1,2,*, Xiao-Jie Ding1,*, Xin Song3, Guang-Biao Zhou4 and Yi Cao11Laboratory of Molecular and Experimental Pathology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China2Kunming College of Life Sciences, University of Chinese Academy of Sciences, Kunming, China3Department of Cancer Biotherapy Center, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province), Kunming, China4State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China*These authors contributed equally to this work
Correspondence to: Yi Cao, email: [email protected] ([email protected])
Keywords: air pollution-related lung cancer; MUC16 (CA125); gene mutation; CRISPR/Cas9 gene editing; biomarker
Received: October 02, 2017 Accepted: December 04, 2017 Published: January 12, 2018Copyright: Kanwal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACTAir pollution is one of the leading causes of lung cancer. Air pollution-related lung
cancer is a deteriorating public health problem, particularly in developing countries. The MUC16 gene is one of the most frequently mutated genes in air pollution-related lung cancer. In the present study, MUC16 mRNA expression was increased in ~50% of air pollution-related lung cancer samples obtained from patients residing in air-polluted regions (Xuanwei and Fuyuan, Yunnan, China), and MUC16 mRNA levels were correlated with the degree of air pollution. Furthermore, sequencing of the captured MUC16 gene identified 561 mutation sites within the MUC16 gene in the air pollution-related lung cancer tissues. Interestingly, some mutations at specific sites and one region were associated with MUC16 mRNA up-regulation. Therefore, we further investigated the impacts of gene mutation on MUC16 expressions and cell behaviors in cultured cells by inducing certain mutations within the MUC16 gene using CRISPER/Cas9 genome editing technology. Certain mutations within the MUC16 gene induced MUC16 overexpression at both the mRNA and the protein level in the cultured cells. Additionally, MUC16 overexpression induced by gene mutations had functional effects on the behavior of lung cancer cells, including increasing their resistance to cisplatin, promoting their growth, and enhancing their migration and invasion capabilities. Based on the data, we suggest that MUC16 mutations potentially associated with air pollution may participate in the development and progression of air pollution-related lung cancer. In addition to ovarian cancer, MUC16 may be a candidate biomarker for lung cancer.
INTRODUCTION
Lung cancer is a leading cause of cancer-related death worldwide. Despite continuous efforts and improvements in the diagnosis and treatment of lung cancer, the overall survival rate is still very low [1]. Additionally, in most patients, lung cancer is already at an advanced stage upon diagnosis, causing single therapy to be mostly ineffective. Improved diagnostics and therapeutics for lung cancer are urgently needed,
and novel tumour biomarkers must be discovered. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer. NSCLC accounts for 80% of all lung cancer cases, including adenocarcinoma (AD), squamous cell carcinoma (SCC), and large-cell carcinoma [2]. Smoking and air pollution are the main causes of lung cancer, and industrial development escalates the levels of air pollution, particularly in developing countries. Air pollution-related lung cancer is a deteriorating public health problem in developing countries [3]. In China, the
Research Paper
Oncotarget12227www.impactjournals.com/oncotarget
rates of lung cancer incidence and mortality have increased rapidly in the past three decades. The lung cancer incidence rates in Xuanwei and Fuyuan in the Yunnan Province of China are among the highest in the country, which is attributed to severe air pollution exposure, specifically to polycyclic aromatic hydrocarbons (PAHs), in these regions [4–6]. The lung cancer cases in these areas are very good models for studying air pollution-related lung cancer [7]. Our previous study determined that mutations in the MUC16 gene were observed in 50% of lung cancer patients residing in Xuanwei and Fuyuan, and the MUC16 gene is among the top frequently mutated genes, thus providing a clue that MUC16 may be associated with air pollution-related lung cancer [6].
MUC16, also named CA125, belongs to mucin family, and mucins are involved in protecting and lubricating epithelial surfaces that line the internal organs of the body. In addition to their normal physiological role in protecting epithelial cells, mucins have been shown to participate in various diseases, including cancer [8]. MUC16, a cell surface glycoprotein with a variable number of tandem repeat structures, was first identified in 1981 [9]. MUC16 is a trans-membrane mucin that was originally detected in epithelial cells and in the mucus layer of the respiratory and gastrointestinal tracts. MUC16, which is cleaved and shed into the bloodstream, is actively researched as a serum biomarker for a variety of tumor types [10]. Greater than 80% of ovarian cancer patients exhibit significantly high MUC16 expression, and CA125 (MUC16) is currently the only serum tumor biomarker routinely used for the clinical diagnosis and predictor of prognosis for ovarian cancer. Additionally, MUC16 is also considered to be a gold standard marker for monitoring ovarian cancer recurrence [11, 12]. Although MUC16 was initially believed to be a specific biomarker of ovarian cancer, MUC16-related studies have clarified that this marker can also be detected in the sera of patients that have other types of cancer, including pancreatic cancer, colorectal cancer, and gastric adenocarcinoma [13, 14].
However, few studies have been conducted to clarify which MUC16 functions boost the development and progression of lung cancer. Additionally, studies regarding the regulatory mechanisms driving abnormal MUC16 gene expression in cancer cells are very limited. Gene mutation is one of main mechanisms underlying gene up-regulation (the gain-of-function) or down-regulation (the loss-of-function). In the present study, we first analyzed MUC16 mRNA expression in lung cancer tissues from patients residing in air-polluted regions (Xuanwei and Fuyuan). We then investigated the impacts of MUC16 gene mutation on MUC16 expression and cell behavior in cultured lung cancer cells by inducing certain mutations within this gene using CRISPR/Cas9 genome editing technology. Our study demonstrated that MUC16 up-regulation induced by gene mutations may be involved in the development and progression of lung cancer and
that MUC16 may be a potential marker for diagnosis, predicting prognosis, monitoring recurrence, and guiding the treatment of NSCLC.
RESULTS
MUC16 mRNA levels in NSCLC tissues were related to air pollution levels
To study the relationship between MUC16 expression and the characteristics of lung cancer patients, we examined the MUC16 mRNA levels in the 84 NSCLC tissues and their adjacent nonmalignant tissues obtained from patients residing in air-polluted regions (Xuanwei and Fuyuan) using qRT-PCR. Compared with those of their matched adjacent noncancerous tissues, the MUC16 mRNA levels were significantly increased in 48.8% (41/84) of the NSCLC tissues (Table 1). This result demonstrates that increased MUC16 expression is associated with cancerous tissue. However, MUC16 mRNA expression did not correlate with gender (p = 0.74), age (p = 0.27), or histology type (p = 0.53). Interestingly, MUC16 mRNA expression was found to be relatively higher in patients living in the heavily and moderately polluted regions of Xuanwei and Fuyuan (p < 0.05, Fisher’s exact test). Though MUC16 up-regulation was observed in 51% of smokers, the overall MUC16 mRNA expression was not significantly different between smokers and non-smokers (p > 0.05, Fisher’s exact test). In addition, statistical analysis concluded that patients who were living in heavily and moderately polluted regions and were also smokers, had higher MUC16 mRNA levels compared to those who were living in relatively clear regions and were non-smokers (p < 0.05, Fisher’s exact test), indicating that air pollution may be the actual cause of MUC16 up-regulation and that smoking could only boost MUC16 expression in the patients residing in areas that were highly polluted. However, our data, which must be categorized as a pilot study, need to be confirmed in further studies.
To evaluate MUC16 expression in cultured lung cancer cells, 14 cell lines were analyzed in this study. Three lung cancer cell lines (A549, 801-D, and NCI-H446) expressed higher MUC16 mRNA levels compared to those of immortal human bronchial epithelial cell lines.
MUC16 gene mutations were detected in lung cancer tissues and cell lines
In total, 22 tissue samples (10 pairs of NSCLC and their adjacent nonmalignant tissues as well as two cancerous tissues) and 10 lung cancer cell lines were selected for sequencing of the captured target gene to analyze the distribution of mutations within the MUC16 gene. The tissues samples were divided into two groups, the MUC16 up-regulated group and the
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MUC16 unchanged/down-regulated group, based on the MUC16 mRNA levels. Various types of mutations within the MUC16 gene were observed (Supplementary Figure 2A). Total single-nucleotide polymorphism (SNP) and insertion and deletion (InDel) data were summarized for each individual and then compared between the two groups. After eliminating all the shared SNPs and InDels, distinct patterns were evident for the MUC16 up-regulated group and the MUC16 unchanged/down-regulated group. Some specific sites and regions that had a significantly unbalanced mutation distribution between the two groups were selected for further study.
Of the 12 sets of NSCLC tissues obtained from patients residing in air-polluted regions (Xuanwei and Fuyuan), seven and five cases showed up-regulated MUC16 mRNA and unchanged/down-regulated MUC16 mRNA, respectively. Overall, 561 mutation sites within the MUC16 gene were identified in both groups (the MUC16 mRNA up-regulated group and the MUC16 mRNA unchanged/down-regulated group). In addition to the 400 mutation sites that were evenly shared among the two groups, 97 and 64 mutation sites were different in the MUC16 up-regulated group and unchanged/down-regulated group, respectively (Supplementary Table 6). A statistically significant difference in the intron mutation rate was observed between the MUC16 up-regulated and unchanged/down-regulated groups (Supplementary Figure 2B). Certain mutation focal points identified within the MUC16 gene displayed an unbalanced mutation rate between the MUC16 up-regulated group and the MUC16 unchanged/down-regulated group (Figure 1A; Table 2). In region 1 (R1; GRCh37/hg19 coordination: 8973772-
8991939), which covers nine small exons and most of the introns, 42 mutation points were found in the MUC16 up-regulated group, whereas only three mutations were reported in R1 in the MUC16 unchanged/down-regulated group. There were significant differences in the mutation rates in R1 between the MUC16 up-regulated group and the MUC16 unchanged/down-regulated group (p < 0.01, Fisher’s exact test) (Figure 1B). Additionally, six mutations were detected in region 2 (R2; GRCh37/hg19 coordination: 9021885-9024488) which spans two exons and most introns, in the MUC16 unchanged/down-regulated group (p < 0.01, Fisher’s exact test), compared to no mutations in R2 being observed in the MUC16 up-regulated group (Figure 1C). In region 3 (R3; GRCh37/hg19 coordination: 9080357-9092214), which covers some of the promoter region and the first and second exon (Figure 1C), the mutation rates were statistically different between the MUC16 up-regulated group and the MUC16 unchanged/down-regulated group (p < 0.01, Fisher’s exact test). Detailed results from these specific regions are listed in Table 2.
We noticed that non-synonymous mutation within the MUC16 gene were detected more frequently in air pollution-related lung cancer in the present study compared with other lung cancer data from the cBioPortal (http://cbioportal.org) [15]. In our previous study, whole genome sequencing also revealed that some genes whose mutation rates and numbers in the lung cancer of highly polluted regions were significantly higher than in NSCLCs of control regions [6]. In addition, non-synonymous mutations were frequently observed in the peripheral blood cell DNA of familial lung cancer samples obtained from
Table 1: Association of MUC16 mRNA expression with clinical and environmental featuresFeatures Total No. (Pairs) MUC16 up-regulated patients Total Subject 84 41 (49%)GenderMaleFemale
5133
26 (50%)15 (45%)
Age (years)����> 60
2658
15 (58%)26 (45%)
Air pollution degreesABC
411924
26 (63%)*11 (57%)*4 (17%)
Smoking statusEverNever
4341
22 (51%) 19 (46%)
Histological typesADSCC
5430
25 (46%)16 (53%)
*P < 0.05; A: heavily polluted regions; B: moderately polluted regions; C: less polluted regions. AD: adenocarcinoma; SCC: squamous cell carcinoma.
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Table 2: Relationships between MUC16 gene mutations at the specific sites and regions as well as MUC16 mRNA expression
Specific-sites /Regions
Functional areas Mutation types
DbSNP141 Positions Total numbers of mutations* P-value
MUC16 up-regulated tissues (7)^
MUC16 unchanged/ down-regulated
tissues (5)^S1 Intron SNP rs62120176 8994784 51 0 0.028
S2 Intron SNP rs55650349 9004587 71 21 0.045S3 Intron SNP rs75371087 9009851 0 31 0.045S4 Intron SNP rs35107941 9013231 51 0 0.028S5 Intron InDel rs34059802 9037305 21 51 0.028S6 Intron InDel Novel 9080281 11 51 0.015S7 Up-stream InDel rs35428697 9092214 0 31 0.045R1 Exon and intron InDel, SNP Novel 8991939-8973772 422 32 0
R2 Exon and intron InDel, SNP Novel 9024488-9021885 0 62 0.002R3 Promoter region and
exonInDel, SNP Novel 9092214-9080357 72 222 0
*Accumulated numbers of mutations for a specific site and a region in the 7 cases of the MUC16 up-regulated tissue or the 5 cases of the MUC16 unchanged/down-regulated tissue; ^Numbers of cases examined in parentheses; S: specific site; R: specific region; SNP: single-nucleotide polymorphism; InDel: insertion and deletion; 1Total numbers of mutation at a specific site. [Note: one mutation may occur at a specific site in a patient]; 2Total numbers of mutations at a specific region. [Note: multiple mutations may occur at a specific region in a patient].
Figure 1: Distribution of mutations within the MUC16 gene. (A) Mutations at specific sites in the MUC16 up-regulated and unchanged/down-regulated groups. (B) More mutations occurred in region 1 (R1) in the MUC16 up-regulated group. (C) Mutations occurring at region 2 (R2) and region 3 (R3) in the MUC16 unchanged/down-regulated group. Statistical significance was calculated using Fisher’s exact test (*p < 0.05, **p < 0.01).
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air-polluted regions (Xuanwei and Fuyuan) in our current study [16]. The reason for these phenomena has not yet been completely explained. We estimate that these may be associated with long-term exposure to high pollution. All of air pollution-related lung cancer patients included in our studies lived in air-polluted areas (Xuanwei and Fuyuan) for more than 30 years.
The statistical relevance between the MUC16 gene mutations and the degree of air pollution was evaluated by calculating p-values. Analysis showed that the heavily polluted areas (A) were linked to higher mutation rates in the intron (Supplementary Figure 2C). These results indicate that air pollution may play an active role in destabilizing the MUC16 gene.
The sequencing data also revealed various mutations in the ten cell lines; however, the mutation distribution patterns were different from those in the tissue samples. Mutations that were identified at several of the specific sites and regions (e.g., S1, S2, S4, S5, and R1) in the lung cancer tissue samples were not prevalent in the cell lines.
MUC16 mRNA was up-regulated after CRISPR/Cas9 gene editing in cultured cells
To further study the relationship between the MUC16 up-regulation and gene mutation, the MUC16 gene was artificially mutated by introducing sgRNA vectors into the genome in cultured cells. One region (R1) and four specific sites (S1, S2, S4, and S5) were selected for this study. A total of 42 mutation points were found in R1 in the seven NSCLC cases in which MUC16 mRNA was up-regulated, but only three mutation points were found in the five cases in which the MUC16 mRNA was unchanged or down-regulated. The mutation frequencies of the four specific sites in the seven NSCLC tissues in which MUC16 mRNA was up-regulated are as follows: 5 cases (S1), 7 (S2), 5 (S4), and 2 (S5). All 14 cell lines were transfected with the two vectors. After screening, the six cell lines with the highest transfection efficiency (293T, A549, 801-D, EPLC-32M1, GLC-82, and SPC-A1) were treated with the seven different mutation systems (S1, S2, S2-1, S4, S5, S5-1, and R1) targeting the four specific sites and one regions, respectively. Overexpressed MUC16 is resistant to cisplatin in ovarian cancer, and cisplatin can be used to select for cisplatin-resistant cell populations [17]. In the present study, cisplatin-resistant cell populations were obtained from the six transfected cell lines after cisplatin treatment with different concentrations (ranging from 0.2-10 µmole/L) and terms of treatment (short- and long-term treatment).
The levels of MUC16 mRNA were verified by qRT-PCR for the seven mutation systems in three different conditions: after transfection alone, after transfection plus short-term cisplatin treatment, and after transfection plus long-term cisplatin treatment. Different mutations within the MUC16 gene resulted in varied expression
patterns; some mutations induced unique alterations in only one cell line, whereas other mutations led to the same alteration in almost every cell line. However, compared to their respective parent cell lines (wild types), MUC16 overexpression was observed in the six selected cell lines for all seven mutation systems (Figure 2). Although the mutation rate at S5 did not statistically correlate with MUC16 mRNA up-regulation in the tissue samples, the cultured cells treated with the S5 and S5-1 systems showed MUC16 mRNA up-regulation. Furthermore, the six cell lines fell into three categories according to their pattern of MUC16 overexpression after transfection and cisplatin treatment: average increase in MUC16 mRNA expression in A549, GLC-82, and EPLC-32M1 cells after transfection alone (without cisplatin treatment); significantly increased MUC16 mRNA expression in SPC-A1 and 801-D cells after transfection plus short-term cisplatin treatment; and significantly increased MUC16 mRNA expression in 293T cells originating from normal human embryonic kidney cells after transfection plus long-term cisplatin treatment. These results indicate that certain mutations at specific foci (S1, S2, S4, and S5) result in MUC16 overexpression in lung cancer cells.
MUC16 protein was up-regulated after CRISPR/Cas9 gene editing in cultured cells
To investigate changes in MUC16 protein expression induced by gene mutations, we performed western blot analysis to semi-quantitatively measure the levels of MUC16 protein in cultured cells after CRISPR/Cas9 gene editing. Western blot analyses of the three cell lines are shown in Figure 3A-C. Like the qRT-PCR results, different mutations showed varied MUC16 protein expression patterns after transfection and cisplatin treatment. Although the MUC16 protein expression was not the same as mRNA expression, overall MUC16 protein levels were elevated in the six cell lines after transfection and cisplatin treatment compared to those of their respective parent cell lines (wild types). We found that mutations at the three foci (S1, S2-1, and S5-1) were related, as similar alterations of MUC16 protein level in the 6 cell lines were observed. The same mutations at these foci were also highlighted for MUC16 mRNA expression, indicating that these mutations may induce MUC16 protein overexpression in cultured cells.
Localization of the MUC16 protein was generally observed at the membrane and in the cytoplasm of cultured cells, as determined by immunofluorescence staining. However, MUC16 was predominantly concentrated in the Golgi apparatus and cytoplasm of EPLC-32M1 parent cells (wild types). Interestingly, MUC16 was more abundant at the membranes and in the cytoplasm of EPLC-32M1 cells after CRISPR/Cas9 gene editing. Moreover, the immunofluorescence signal for MUC16 was strengthened after treatment with almost all the mutation
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focus systems compared to that of their parent cells (wild types) in the six cell lines, validating the western blotting results. Immunofluorescence staining results in the three cell lines is shown in Figure 3D.
The western blot and immunofluorescence analyses confirmed that MUC16 protein levels were increased in cultured cells after CRISPR/Cas9 gene editing, which suggests that certain mutations within the MUC16 gene may have a functionally disruptive impact on MUC16 protein expression. The stably mutated cells obtained using CRISPR/Cas9 gene editing that overexpress MUC16 protein can be used for functional experiments.
MUC16 overexpression induced by CRISPR/Cas9 gene editing stimulated lung cancer cell proliferation
To further investigate the influence of MUC16 gene mutations on cellular behavior, we selected the most important mutations at the three foci (S1, S2-1, and
S5-1), and scrutinized the effects of these mutations on cell proliferation in A549, EPLC-32M1, and 293T cells. Significantly increased proliferative ability was observed in the three cell lines subjected to CRISPR/Cas9 gene editing compared with the abilities of their respective parent cells (wild types) (Figure 4A), particularly in the A549 and EPLC-32M1 cells (representative lung cancer cell lines). These results reveal that MUC16 overexpression induced by gene mutations may promote lung cancer cell growth.
MUC16 overexpression induced by CRISPR/Cas9 gene editing was associated with resistance to cisplatin in lung cancer cells
In the present study, the MTT assay was used to test the sensitivity of anti-cancer drugs in A549, EPLC-32M1, and 293T cells. In general, the transfected cells exhibited a higher tolerance to cisplatin compared with that of their parent cells (wild types). As expected, the
Figure 2: MUC16 mRNA expression in cultured cells after MUC16 gene editing. (A–F) Increased MUC16 mRNA levels were observed in the six cell lines after transfection alone (transfection), transfection plus short-term cisplatin treatment (Short cis treatment), and transfection plus long-term cisplatin treatment (Long cis treatment) compared to those of the parent cell lines (wild types). Cis: cisplatin. S1, S2, S2-1, S4, S5, S5-1, and R1: mutation systems used in this study.
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cisplatin-resistant cell populations showed a stronger tolerance to cisplatin. For example, the IC50 of cisplatin in the cisplatin-resistant A549 line was approximately 23.20 ± 32.97 µM (short-term treatment), which was 3–4-fold higher than that of its parent line (7.27 µM), and the IC50 of cisplatin in the cisplatin-resistant EPLC-32M1 line was 10.38 ± 29.64 µM (long-term treatment), which was 2–7-fold higher than that of its parent line
(4.66 µM). Resistance curves depicted slightly increased cisplatin survival ability in A549 and EPLC-32M1 cells (Figure 4B). These results demonstrate that MUC16 overexpression induced by gene mutations may develop resistance to cisplatin in lung cancer cells.
In the present study, the cisplatin-resistant cell populations were exposed to azacitidine and paclitaxel, to which they were also sensitive (Supplementary Figure 3),
Figure 3: MUC16 protein expression in cultured cells after MUC16 gene editing. (A–C) Western blot analysis of MUC16 protein levels in the three cell lines after transfection alone (Transfection), transfection plus short-term cisplatin treatment (Short cis treatment), and transfection plus long-term cisplatin treatment (Long cis treatment). Compared to those of the parent cell lines (P and Parent), elevated protein levels were observed in cultured cells after MUC16 gene editing. The MUC16 protein band corresponding to 180 kDa (left) ZDV�XVHG�IRU�VHPL�TXDQWLWDWLYH�DQDOVLV��Į�7XEXOLQ�����N'D��ZDV�XVHG�DV�WKH�LQWHUQDO�FRQWURO�DQG�UHIHUHQFH�IRU�VHPL�TXDQWLWDWLYH�DQDOVLV��(D) MUC16 protein was stained in the three cell lines using immunofluorescent antibodies. The staining signal was slightly increased at the membranes and in the cytoplasm of A549 and 293T cells after treatment compared to that of the parent cells. Note: the staining was obviously enhanced at the membranes of EPLC-32M1 cells after treatment. Parent: parent cells (wild types); Trans: transfection alone; Cis: transfection plus long-term cisplatin treatment. S1, S2, S2-1, S4, S5, S5-1, and R1: mutation systems used in this study.
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indicating that cisplatin-resistant lung cancer cells are not tolerant towards other anti-cancer drugs, such as azacitidine and paclitaxel.
MUC16 overexpression induced by CRISPR/Cas9 gene editing promoted the migration and invasion capacity of lung cancer cells
To further gain insight into whether the dissemination of lung cancer cells is related to MUC16 overexpression and gene mutation, cellular migration and invasion were evaluated in A549 and EPLC-32M1 cells after they were transfected with the three mutation foci (S1, S2-1, and S5-1) and treated with cisplatin. Compared to those of the parent cells (wild types), MUC16 overexpression induced by gene mutations significantly enhanced the migration and invasion efficiency of A549 and EPLC-32M1 cells (Figure 5). The ability of cell to migrate and invade is closely associated with the invasion and metastasis of cancer cells. Taken together, these findings indicate that MUC16 overexpression induced by gene mutations may promote the invasion and metastasis of lung cancer.
DISCUSSION
MUC16 is a trans-membrane glycoprotein that efficiently modulates cell adhesion, protein-protein interaction, and immunity by altering its expression and the nature of glycosylation [11, 18]. Our previous study demonstrated that 50% of air pollution-related lung cancers contain a mutated MUC16 gene [6]. In a general study of lung cancer, 53% (302/572) of lung cancers, including 51% (202/394) of AD and 56% (100/178) of SCC, showed mutations in the MUC16 gene [19]. MUC16 was one of three genes having the highest mutation frequency across multiple cancer types [19, 20]. The high mutation frequency of MUC16 gene was largely due to its long sequence in some cancers such as breast, liver, kidney cancer. After correcting for sequence length, MUC16 was not ranked in the top 10 of mutated genes for these cancers. However, MUC16 was still retained in the top 10 of mutated genes for lung and large intestine cancer after correcting for sequence length [20]. In the present study, we investigated the mRNA levels of MUC16 in lung cancer tissue samples obtained from patients residing in air-polluted regions (Xuanwei and Fuyuan) and observed
Figure 4: Cellular proliferation and resistance to cisplatin in cultured cells after MUC16 gene editing. (A) The bar graphs demonstrate a significant increase in the proliferation rate of cultured cells after treatment compared to that of the respective parent cells (Student’s t-test; *p < 0.05, **p < 0.01). (B) Parent cells, transfected cells, and cisplatin-resistant cell populations were incubated with different concentrations of cisplatin, and then cell viability was determined. The cell-survival curves demonstrate a significant increase in cisplatin resistance in the cultured cells after treatment compared to that of the respective parent cells. Parent: parent cells (wild types); S1, S2-1, and S5-1: mutation systems used in this study; Trans: transfection alone; Short Cis: transfection plus short-term cisplatin treatment; Long Cis: transfection plus long-term cisplatin treatment.
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that MUC16 mRNA was up-regulated in 48.8% (41/84) of the cancerous tissue samples compared to that of their adjacent normal tissues (p < 0.05). This suggests that MUC16 mRNA up-regulation is associated with cancerous tissues. Furthermore, statistical analysis concluded that MUC16 mRNA expression was correlated with the degree of air pollution, and patients living in heavily and moderately polluted regions who were also smokers, showed significantly increased MUC16 mRNA expression compared to those who were living in comparatively clean areas and were non-smokers (p < 0.05).
In addition, we sequenced DNA from captured MUC16 genes in air pollution-related lung cancer tissue samples. Sequencing data indicated that the mutation rate of the intron within the MUC16 gene was significantly related to the degree of air pollution (p < 0.05). Several studies have reported that lung cancer in the Xuanwei population is associated with air pollution, particularly air polluted with PAHs [4–6]. PAHs, burning products of tobacco, gasoline, diesel, coal, et cetera, are the most important carcinogens resulting from smoking and air pollution. Because BaP is the major PAH index,
the BaP concentration in the polluted air represented the degree of air pollution in Xuanwei and Fuyuan in this study. PAHs can directly bind DNA, form DNA adducts, and induce gene mutation [5, 21], which can result in up-regulation or down-regulation of the gene. Our results demonstrated that MUC16 up-regulation and gene mutation occurred simultaneously in air pollution-related lung cancer, and MUC16 up-regulation and mutation were associated with the degree of air pollution or, more precisely, the degree of PAH exposure. Moreover, PAHs DUH�DVVRFLDWHG�ZLWK�&�*ĺ$�7�WUDQVYHUVLRQV�>������@��,Q� RXU� SUHYLRXV� VWXG�� &�*ĺ$�7� WUDQVYHUVLRQV� ZHUH�the most frequent nucleotide substitution in lung cancer patients residing in Xuanwei and Fuyuan, as determined by whole-genome sequencing and exome sequencing >�@��,Q�WKH�SUHVHQW�VWXG��&�*ĺ$�7�WUDQVYHUVLRQV�ZHUH�also observed in the mutated MUC16 gene (data not shown). Based on these data, we hypothesized that air pollution and PAH exposure may cause MUC16 gene mutations, which can subsequently lead to changes in MUC16 mRNA expression in air pollution-related lung cancer.
Figure 5: Cultured lung cancer cell invasion and migration after MUC16 gene editing. (A) Cell invasion and migration assays in A549 and EPLC-32M1 cells after treatment. Invading and migrating cells were photographed. (B) Results of the treated cells were statistically analyzed and compared with those of the parent cells. The fold changes in the numbers of invading and migrating cells are given as the means ± SD from three independent experiments (Student’s t-test; *p < 0.05). Parent: parent cells (wild types); S1, S2-1, and S5-1: mutation systems used in this study; Tran: transfection alone; Long Cis: transfection plus long-term cisplatin treatment.
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Interestingly, sequencing data revealed that some mutations at specific sites and regions were associated with MUC16 mRNA up-regulation. To confirm that MUC16 gene mutations could induce MUC16 overexpression, we focused on mutations at four specific sites (S1, S2, S4, and S5) and one region (R1) that occurred more in lung cancer tissues in which MUC16 mRNA was up-regulated. The CRISPR/Cas9 genome editing technique was employed to insert mutations in the six selected cell lines (A549, EPLC-32M1, GLC-82, SPC-A1, 801-D, and 293T) using seven different mutation systems (S1, S2, S2-1, S4, S5, S5-1, and R1). In general, MUC16 expression was enhanced at both the mRNA and protein level in the cultured cells after certain mutations were induced within the MUC16 gene using CRISPR/Cas9. Because cancer cells in which MUC16 was overexpressed have been shown to be resistant to cisplatin in ovarian cancer [14], we also treated the cultured cells with cisplatin after the MUC16 gene was edited, and MUC16 overexpression induced resistance to cisplatin in the lung cancer cells. However, no significant changes in the cell-survival curves were observed after treating the cisplatin-resistant cell population with azacitidine and paclitaxel, indicating that the cisplatin-resistant cell populations still show sensitivity to azacitidine and paclitaxel. Based on the targeted gene mutation in vitro experiments, we proposed that certain mutations within the MUC16 gene could induce MUC16 overexpression and that lung cancer cells in which MUC16 overexpression was induced by gene mutations could show resistance to cisplatin in a manner similar to ovarian cancer cells.
Finally, we investigated the effects of MUC16 overexpression induced by gene mutations on cell growth, migration, and invasion in cultured lung cancer cells. Cellular proliferation, migration and invasion abilities were significantly increased by MUC16 overexpression induced by mutations in the MUC16 gene. Thus, we suggest that MUC16 overexpression induced by gene mutations have functional impacts on cell behaviors, such as proliferation, migration, and invasion, in lung cancer. Cellular proliferation, migration, and invasion play active roles in the development and progression of cancer. Analogously, a recent study has also reported a strong association between increased MUC16 expression and aggressiveness of cisplatin resistant lung cancer cells. Through the JAK2/STAT3/GR axis, MUC16 overexpression down-regulates TSPYL5, which further mediates chemoresistance, proliferation and metastasis of lung cancer cells by suppressing p53 [24]. Additionally, although MUC16c354 transgenic animals have a normal lifespan, spontaneous tumors (including carcinoma in lung) arise at higher frequency in the double MUC16c354:p53+/- mice compared to p53+/- mice alone [25]. In previous studies, MUC16 up-regulation was shown to contribute to the invasion, aggression, and metastasis of tumor cells in various cancer types [12, 13, 26],
particularly in ovarian cancer [27]. Moreover, MUC16 that is shed into the bloodstream can bind to certain cell types, such as natural killer (NK) cells and monocytes, to induce functional responses [18]. MUC16 up-regulation may protect cancer cells from the immune response and prevent cancer cells from cytolysis [28, 29]. Furthermore, MUC16 up-regulation has been shown to correlate with ovarian cancer relapse [30] and poor prognosis [31]. In addition to ovarian cancer, MUC16 up-regulation may be involved in the development and progression of pancreatic cancer [26]. Based on previous studies and our present study, we speculate that MUC16 gene overexpression induced by gene mutations is not only a phenomenon, but also plays a functional role in the development and progression of lung cancer.
In conclusion, high concentrations of carcinogens in polluted air may be associated with MUC16 gene mutations, and certain mutations (not all mutations) within the MUC16 gene can induce MUC16 overexpression at both the mRNA and protein level. Additionally, MUC16 overexpression has functional impacts on the behavior of lung cancer cells, including increasing their cisplatin resistance, promoting their growth, and enhancing their migration and invasion capabilities. Thus, MUC16 mutations potentially caused by air pollution may participate in the development and progression of air pollution-related lung cancer. In addition to ovarian cancer, MUC16 may be a candidate biomarker for air pollution-related lung cancer.
MATERIALS AND METHODS
Tissue samples and cell lines
A total of 84 pairs of lung cancer tissue samples were collected during surgery, which included both cancerous tissues and adjacent nonmalignant lung tissues from the same patient. Two pathologists confirmed the histological characteristics of the tumors based on their World Health Organization classification [32]. The tumor tissues that were > 70% cancer and < 10% necrotic were selected for this study, and the matched adjacent tissues contained no cancer cells. The 84 lung cancer samples comprised 54 AD cases and 30 SCC cases. The patients were between 29 and 76 years of age (average age, 52.5 years) and included 51 men and 33 women. All patients lived in Xuanwei City and Fuyuan County in Yunnan Province, China. Similar to previous studies [5, 6, 33], the entire area was further divided into three regions, heavily polluted, moderately polluted, and less polluted, according to the concentration of indoor and outdoor benzo(a)pyrene (BaP), which is the major index of PAH carcinogenicity (Supplementary Figure 1). Patient information is listed in Supplementary Table 1. This study was approved by the Ethics Committee for Human Medicine Research at Yunnan Tumor Hospital and the Kunming Institute of Zoology at the Chinese Academy of Sciences.
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Fourteen cell lines (eleven lung cancer cell lines, two immortal human bronchial epithelial cell lines, and one human embryonic kidney cell line) were included in the study. The cells were cultured in either RPMI-1640 or DMEM supplemented with 10% fetal bovine serum (FBS) according to a standard protocol and maintained in an incubator at 37°C with 5% CO2. The cell line information is provided in Supplementary Table 2.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from frozen tissue samples and cultured cells using TRIzol (Invitrogen, Carlsbad, CA, USA) and the phenol-chloroform extraction standard method, and cDNA was synthesized using a reverse-transcription kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. RT-PCR was performed using the StepOne Real-time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR Green (Invitrogen). The level of MUC16 mRNA was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the comparative threshold cycle number �&W��PHWKRG�ZKHUH�¨¨&W= ¨&W �7DUJHW����¨&W��&RQWURO���Changes in the MUC16 mRNA levels (up-regulated, down-regulated, or unchanged) were determined using the 2�¨¨&W method as described by Livak [34]. The amplification products were analyzed using melting curve analysis, and all the reactions were repeated three times to confirm the results. The primers are: MUC16-F ��MUC16-R�މ��*&&$&$&&$&**$&$$&&&&7*�މ����މ��77$7*77*&$**7&*77*7&$&***�މ�� >��@��*$3'+�)� ���މ��77&&&&$*7$$&7$&&*77*�7މ����މ��*&*$&7&$7*&$*&$&&7&�މ����5+'$3*
Western blot analysis
Cells were lysed in RIPA buffer (Solarbio, Beijing, China) supplemented with protease inhibitors (Sigma, St. Louis, MO, USA). Proteins were subjected to 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After being blocked with 5% bovine serum albumin (BSA), the membranes were probed IRU� 08&���� Į�WXEXOLQ� ZDV� XVHG� DV� D� ORDGLQJ� FRQWURO��Mouse anti-human MUC16 monoclonal antibody (mAb) (stock: 764 µg/ml dilution factor 1:1000) and mouse DQWL�KXPDQ�Į�WXEXOLQ�P$E��GLOXWHG����������6DQWD�&UX]�Biotechnology, Santa Cruz, CA, USA) were the primary antibodies used. After being incubated with peroxidase-labelled goat anti-mouse IgG [H+L] (Kirkegaard & Perry Lab, Gaithersburg, MD, USA), the proteins were detected using a chemiluminescent peroxidase substrate (Millipore). The blot X-ray films were processed with ImageJ software (NIH, Bethesda, MD, USA) for semi-quantitative measurements.
Immunofluorescence staining
Cells were fixed with methanol and washed with phosphate buffered saline (PBS). After being blocked with 1% BSA, the cells were incubated with the primary antibody (Stock: 764 µg/ml; dilution factor 1:500) (mouse anti-human MUC16 mAb; Santa Cruz Biotechnology) at 4°C overnight. The next day, the cells were washed and incubated with two secondary antibodies (dilution factor 1:1000) (FITC/Cy3-labeled goat anti-mouse IgG; Kirkegaard & Perry Lab) separately to detect both green and red fluorescent signals. The nuclei were counterstained with Hoechst 33258 (Sigma). Images were acquired with a confocal microscope (Nikon, Tokyo, Japan).
Target gene sequencing and specific mutation sites selection
Genomic DNA samples from tissues (ten pairs of lung cancer tissues and their adjacent nonmalignant tissues as well as two cancerous tissues) and ten cancer cell lines (all lung cancer cells except for the 95-D line) were used for sequencing of the captured target gene. Genomic DNA was prepared according to the guidelines of the International Cancer Genome Consortium (http://www.icgc.org/policies). MUC16 gene capture and sequencing were performed by the BGI-Tech Company (Shenzhen China; http://www.genomics.cn). The total DNA available for all samples was fragmented to an average insert size of 145 bp (range 75–300 bp) and subjected to Illumina DNA sequencing library preparation. The MUC16 gene was captured using the Agilent Sure Select Capture System (Agilent Technologies, Santa Clara, CA, USA), and DNA sequencing was performed using the Illumina HiSeq 2000/2500 platform. Each sample was sequenced at the mean depth of 140× (54.8× – 261×) coverage. Sequencing reads were aligned to the NCBI-built 37 human genome. Somatic mutations of the MUC16 gene were identified through standard bioinformatics (in silico) analyses by the BGI-Tech Company. Data analysis and sequence alignment was performed to point out all the novel and known mutation in the MUC16 gene by using different online tools and software followed by statistical analysis.
Vector construction for editing the MUC16 gene
CRISPR/Cas9 vectors were constructed for targeting the selected specific sites and regions within the MUC16 gene. All sgRNAs were designed using CRISPRdirect (http://crispr.dbcls.jp/). The sgRNA oligomers were synthesized and cloned into the pSpCas9(BB)-2A-GFP (PX458) vector (Addgene plasmid ID 48138). The sgRNAs were cloned by annealing two DNA oligos and ligating into a BbiI-digested vectors as previously described [35]. To improve the promoter efficiency, ZH�DGGHG�DQ�H[WUDމ���*�QXFOHRWLGH�WR�DOO�RI�WKH�VJ51$V�
Oncotarget12237www.impactjournals.com/oncotarget
WKDW�GLG�QRW�VWDUW�ZLWK�D��7*�މ��KH�SODVPLG�FRQWDLQLQJ�CRISPR/Cas9 target site for each target mutation was confirmed by plasmid DNA sequencing. A mixture of 1 ȝJ�RI�3;����SODVPLG�'1$�FRQWDLQLQJ�HDFK�WDUJHW�VJ51$�sequence was used for cultured cell transfection. A total of 17 sgRNA vectors targeting four specific sites and one region were built. All the sgRNA sequences are presented in Supplementary Table 3. All 17 vectors were further divided and organized into seven different groups: four specific sites (S1, S2, S4, and S5) each targeted by one vector that induced a single cut at the site (including four vectors); two specific sites (S2-1 and S5-1) each targeted by two different vectors that made double cuts upstream and downstream of the site (deleted 50-260 bp; including four vectors); and region 1 (R1) targeted by nine vectors that caused multiple cuts spanning 18 kb.
Vector transfection
Cultured cells were first seeded in 6-well plates and then transfected using Lipofectamine 2000 (Invitrogen) when they reached a density of 70% following the manufacturer’s instructions. Two plasmids (pSpCas9(BB)-2A-GFP and Phage-to-dCas9-3XmCherry expressing green and red fluorescence, respectively) were used to evaluate transfection efficiency. Each cell line was transfected with the two plasmids separately. After the cells were incubated for an additional 48 hours (hr), they were analyzed on a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). All 14 cell lines transfected with vectors were screened to select for cell lines that had a high transfection rate. The empty vectors were used as an internal control.
Selection of cisplatin-resistant cell populations
Cancer cells that overexpress MUC16 are resistant to cisplatin in ovarian cancer, and cisplatin has been used for MUC16-selective modulation [12]. Thus, after the selected cell lines were transfected with the constructed vectors of the seven different groups, they were maintained in culture medium containing cisplatin (Sigma) to obtain cisplatin-resistant cell populations for further study. Through cisplatin treatment, the transfected cell populations that expressed MUC16 survived, and the untransfected cell populations that did not express MUC16 were killed by cisplatin. The treatment details, including dosage and time, are listed in Supplementary Table 4. After being selected, the cells were cultured without cisplatin.
Cell proliferation and cytotoxicity assay
Cell proliferation was analyzed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After 24, 48 and 72 hr of treatment, the MTT reagent (Sigma) was added, and the mixture was incubated
for 3 hr at 37°C. Dimethyl sulfoxide was then added, and the absorbance was measured at 595 nm by a microplate reader (Bio-Rad, Hercules, CA, USA).
Cisplatin, azacitidine, and paclitaxel were purchased from Sigma and dissolved in 0.1 M NaCl solution. The aliquots were stored at –20°C and thawed immediately prior to use. For the cytotoxicity assay, cells were seeded at a density of 20,000 cells per well in 96-well plates. 7KH�QH[W�GD��¿[HG�GRVHV�RI�WKH�GUXJV�ZHUH�DGGHG��DQG�the mixtures were incubated for an additional 72 hr. The drug concentrations are listed in Supplementary Table 5. Cell viability was estimated by the MTT assay, and the SHUFHQWDJH�RI�FHOO�VXUYLYDO�ZDV�GH¿QHG�DV� WKH�UHODWLYH�absorbance of the treated cells versus the untreated cells. All assays were repeated three times.
Migration and invasion assay
For the migration assay, trans-well inserts (pore VL]H����ȝP��0LOOLSRUH��ZHUH�ILUVW�LQFXEDWHG�DW����&�LQ�D�CO2 incubator for 1 hr. For the invasion assay, the same inserts were first coated with Matrigel (BD Bioscience). Next, either DMEM or RPMI 1640 supplemented with 10% FBS was added into the lower chamber, and cells in serum-free DMEM or RPMI 1640 were placed into the upper chamber. The cells were allowed to migrate for 24 hr, and cells on the inserts were then fixed with methanol and stained with crystal violet. The non-migrated cells on the upper side of the chamber were removed. The insert membranes were scanned and analyzed using NIH image software (https://imagej.nih.gov/ij/), and the cell density is expressed as pixel intensity.
Statistical analysis
All statistical analyses were conducted using SPSS 17 software (SPSS, Chicago, IL, USA). Measurement data were evaluated by the Student’s t-test, and enumeration data were analyzed using the chi-squared test or the Fisher’s exact test. 'LIIHUHQFHV�ZHUH�FRQVLGHUHG�VLJQL¿FDQW�DW�*p < 0.05; **p < 0.01.
ACKNOWLEDGMENTS AND FUNDING
This study was supported by the Natural Science Foundation of China (81272617), the 973 Program (2011CB510104) and the Yunnan Province Science and Technology Department (Y103951111), and sponsored by CAS-TWAS President’s Fellowship for International PhD Student (to MK). We thank Dr. P. Wang (Department of Surgery, Yunnan First People’s Hospital) for providing the surgical samples.
CONFLICTS OF INTEREST
Authors declared no conflicts of interest relevant to this study.
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http://dx.doi.org/10.2147/OTT.S163774
NEAT1_2–SFPQ axis mediates cisplatin resistance in liver cancer cells in vitro
Yi Ru1–4
Xiao-Jie Chen5
Wen-Zhi Guo1–4
She-Gan Gao5
Yi-Jun Qi5
Pan Chen5
Xiao-Shan Feng5
Shui-Jun Zhang1–4
1Henan Key Laboratory of Digestive Organ Transplantation, 2Open Laboratory of Key Disciplines of Hepatobiliary and Pancreatic Surgery and Digestive Organ Transplantation, 3Key Laboratory of Hepatobiliary and Pancreatic Diseases and Organ Transplant Medicine, 4Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, People’s Republic of China; 5Henan Key Laboratory of Cancer Epigenetics; Cancer Institute, The First Affiliated Hospital and College of Clinical Medicine of Henan University of Science and Technology, Luoyang, Henan Province, People’s Republic of China
Background: Liver cancer is a type of malignant tumor with high morbidity and mortality in People’s Republic of China. Its occurrence and development involve the variation and expression changes of multiple genes, and the pathogenesis and related regulatory networks are complex.Purpose: In the present research, we investigate the involvement of NEAT1_2 and SFPQ in cisplatin resistance in liver cancer. The effects of LncRNA NEAT1 and SFPQ expression on the chemotherapeutic resistance of liver cancer cells were analyzed.Methods: The expression level of NEAT1_2 and SFPQ mRNA in tissue specimens or cell lines were examined by RT-qPCR and western blotting. CCK-8 assay was performed to evaluate cell viability. Cell proliferation was performed using the EdU cell proliferation assay.Results: Our data showed that increase NEAT1_2 and SFPQ expressions in liver cancer speci-mens were associated with the development of cisplatin resistance; high SFPQ expression level impaired patients’ survival from liver cancer. Gain-and loss-of function assay using NEAT1_2 knock-in and knock-out cells constructed using CRISPER/Cas9 system revealed that NEAT1_2 is essential for liver cancer cell survival and mediates cisplatin resistance in liver cancer cells at least partially through SFPQ. Artificial change in NEAT1_2 expression level didn’t significantly influence SFPQ transcription or translation level.Conclusion: Our data revealed NEAT1_2—SFPQ axis as a novel cisplatin resistance mecha-nism in liver cancer cells in vitro.Keywords: NEAT1, NEAT1_1, NEAT1_2, SFPQ, liver cancer, cisplatin resistance
IntroductionLiver cancer is the second leading cause of cancer related death in People’s Republic of China.1 Chemotherapy of different approaches using platinum drugs is seemingly an optimal strategy for treating late stage liver cancer,2,3 but primary or acquired platinum drug resistance remains a major obstacle in clinic practice. Alteration in the nucleotide excision repair (NER) pathway plays an important part in the development of platinum drug resistance, and the expression level of NER-related proteins has been proposed as potential biomarker for poor response to platinum-based chemotherapy.4,5 Briefly, platinum drugs exhibit anti-cancer effects by inducing DNA damage via forming a DNA adduct with inter- or intra-strand crosslinks between guanine bases, thus distort-ing the DNA helix, inhibiting DNA replication, eliciting DNA damage response/repair mechanisms in cells, and eventually activating the apoptosis program when the damage is unrepairable. The DNA damage response process in living cells includes cell cycle arrest, damage repair, and cell cycle restart. DNA helix distortion is caused by platinum drug-induced DNA damage triggering the activation of NER system,6 which removes the DNA lesions caused by platinum drugs via sensing and excision of the lesion fol-lowed by DNA re-synthesis and ligation.7 In platinum drug sensitive cells, the prolonged
Correspondence: Shui-Jun ZhangDepartment of Hepatobiliary and 3DQFUHDWLF�6XUJHU��)LUVW�$IÀOLDWHG�Hospital of Zhengzhou University, 1 Jian-she Road, Er-qi District, Zhengzhou, Henan 450052, People’s Republic of ChinaEmail [email protected]
Xiao-Shan FengHenan Key Laboratory of Cancer Epigenetics, Cancer Institute, The )LUVW�$IÀOLDWHG�+RVSLWDO�DQG�&ROOHJH�RI�Clinical Medicine of Henan University of Science and Technology, 24 Jing-hua Road, Luoyang, Henan 471003, People’s Republic of ChinaEmail [email protected]
Journal name: OncoTargets and TherapyArticle Designation: Original ResearchYear: 2018Volume: 11Running head verso: Ru et alRunning head recto: NEAT1_2!SFPQ mediates liver cancer cisplatin resistanceDOI: 163774
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Ru et al
DNA damage repair process leads to P53-dependent and independent apoptosis, while those cells with hyper-activated NER machinery or overexpressed NER proteins often show resistance to platinum drug-induced cytotoxity.8–12
NEAT1 is an lncRNA that includes two transcript sub-types, NEAT1_1 and NEAT1_2. NEAT1_1 and NEAT1_2 are 3.7 kb and 23 kb in length, respectively. NEAT1_1 is part of NEAT1_2. NEAT1_1 and NEAT1_2 have a 3.7 kb overlap at the 5′ end. One of our preliminary studies screened tran-script abundance changes associated with cisplatin resistance and suggested that a nuclear associated lncRNA, NEAT1_2, might be involved in the development of platinum drug resis-tance in liver cancer cells.17 The cancer-promoting role of NEAT1 in different cancer types, including liver cancer, with its considerable prognostic value, has been revealed by differ-ent studies,13–17 the molecular mechanism of which, however, remains largely unresolved. Despite the increasing focus on NEAT1 regulating microRNA expression, a recent report by Adriaens et al raised the possibility that NEAT1_2 encour-ages platinum drug resistance by reducing the accumulation of DNA damage and replication stress-induced cell death.18
As a key component in paraspeckle, a subnuclear struc-ture involved in regulating gene expression, stress response, and cell cycle,19,20 NEAT1_2 interacts with three out of five discovered paraspeckle proteins, namely PSPC1, SFPQ, and NONO, all of which belong to the DBHS protein family and are fundamentally and dynamically dimerized.21,22 The involvement of PSPC1 and NONO in platinum drug resis-tance has been reported,23,24 but the role of SFPQ in this pathophysiological development remains undetermined. By constructing NEAT1_2 knock-out cell lines with or with-out SFPQ RNA interference, we verified the involvement of both NEAT1_2 and SFPQ in the development of cisplatin resistance in liver cancer cells. We hope the results of the present research could provide some new insight into the molecular mechanism of platinum drug resistance.
Materials and methodsLiver cancer patients and tissue specimensThis research was approved by the medical ethics committee of the First Affiliated Hospital of Zhengzhou University. Five patients diagnosed with primary liver cancer on the first visit were enrolled in the naïve group, compared to five patients enrolled in the cisplatin-resistant group who bore cisplatin-resistant primary liver cancers. Written informed consent was obtained from each patient before enrollment. All patients’ primary liver cancers were diagnosed following a practical standard in reference to American Association for the Study of Liver Diseases criteria. Patients’ liver cancer pathologic
tissue specimens and non-pathologic adjacent tissue speci-mens were obtained by aspiration biopsy for therapeutic purposes. Determination of cisplatin-resistant patients: in patients with liver cancer after the first and second cycles of cisplatin chemotherapy, the tumor was relieved; however, in the third and fourth cycles, tumor remission was not obvi-ous and progressed, with some side effects, such as nausea, vomiting, liver area pain, low fever, hair loss, hepatomegaly; some patients had ascites in the abdomen. The CT results showed that the cancer did not shrink, and the blood AFP increased again.
RT-qPCR and Western blottingRT-qPCR of NEAT1_2 and SFPQ mRNA in tissue speci-mens or cell lines were performed using a custom-made RT-qPCR kit (GeneCopoeia, Maryland, USA), following the manufacturer’s instructions. The semi-quantitative 2−ΔΔCt method was employed for qPCR data analysis, and expression levels of NEAT1_1, NEAT1_2 or SFPQ mRNA in each sample were normalized to that of GAPDH mRNA before further analysis. Western blotting of SFPQ in differ-ent cell cultures was performed using a rabbit anti-human SFPQ monoclonal antibody (ab177149), rabbit anti-human GAPDH monoclonal antibody (ab128915) and horseradish peroxidase-conjugated goat anti-rabbit second antibody (ab205718, Abcam, Cambridge, UK), following the manu-facturer’s instructions. Western blotting results were further analyzed by normalizing the gray scale of the SFPQ band in each sample to that of GAPDH using ImageJ software before statistical analysis.
Cell culture and preparationThe six liver cancer cells used in this research were previ-ously purchased from the American Type Culture Collec-tion and maintained in liquid nitrogen before use. Cells were cultured in DMEM medium supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified sterile cell culture incubator with 37°C, 5% CO
2,
100% humidity atmosphere. NEAT1 knock-out in QGY-7703 liver cancer cells at log-phase was performed using a custom-made Cas9-sgRNA NEAT1 knock-out kit (GeneCo-poeia), following the manufacturer’s instructions. Briefly, vectors loaded with Cas9 gene and sgRNA targeting the promoter sequence of NEAT1 were transfected into QGY-7703 cells, before single cell clones were isolated by serial dilutions. NEAT1 knock-out clones were selected using the IndelCheck™ kit provided with the NEAT1 knock-out kit, following the manufacturer’s instructions. NEAT1 knock-in in HUH-7 cells was performed using a custom-made AAVS1
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NEAT1_2–SFPQ mediates liver cancer cisplatin resistance
safe harbor gene knock-in kit (GeneCopoeia), following the manufacturer’s instructions. Briefly, AAVS1 sgRNA/Cas9 expression vector and NEAT1 donor vector were co-trans-fected into HUH-7 cells. Positive knock-in cells were selected by neomycin screening. Transient SFPQ gene silencing was performed by shRNA targeting, using a custom-made SFPQ knockdown kit (GeneCopoeia), following the manufacturer’s instructions. Gene editing of NEAT1 or SFPQ in each cell line was verified by RT-PCR before being subjected to this research (data not shown).
Cell function assaysThe CCK-8 assay was performed to evaluate cell viability, using CCK-8 reagent purchased from Beyotime (Shanghai, People’s Republic of China), following the manufacturer’s instructions. OD450 of each cell culture after addition of CCK-8 reagent was measured at different time points with a microplate reader. Annexin V/propidium iodide double staining was used for apoptosis assay, using a dead cell apoptosis kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Cell staining of fluorescent conjugated Annexin V and propidium iodide was measured by flow cytometry. The cell proliferation assay was performed using an EdU cell proliferation detection kit (Sigma-Aldrich, St Louis, MO, USA), following the manu-facturer’s instructions. Fluorescent EdU and DAPI double staining of each cell culture were evaluated microscopically. Cell proliferation rate was calculated by comparing EdU posi-tively stained cells over all DAPI positively stained cells.
Statistical analysisAll statistical analyses and visualization in this research were performed using GraphPad Prism 7 software. Unless indi-cated otherwise, all data is presented as mean ± SD; Student’s t-test was used to test for significance between groups, and a P,0.05 was considered statistically significant.
ResultsHigh NEAT1_2 and SFPQ expression correlates with cisplatin resistance and liver cancer developmentWe first evaluated NEAT1_1, NEAT1_2 and SFPQ expres-sion levels in paired liver cancer tissue specimens and non-cancerous adjacent tissue specimens obtained from five liver cancer patients who were diagnosed on the first visit (naïve) or five patients who had developed cisplatin resistance. NEAT1_1 expression increased in cancerous tissue specimens comparing to non-cancerous adjacent tissue specimens but was 2- to 3-fold lower than that of NEAT1_2,
while no significant difference between specimens from first naïve patients and cisplatin resistance patients was observed (data not shown), which implied that NEAT1_1 or its upregulation might not be imperative for the develop-ment of cisplatin resistance in liver cancer. Transcriptional levels of both NEAT1_2 and SFPQ were also significantly increased in liver cancer tissue specimens, compared to non-cancerous adjacent tissue specimens, but were further upregulated in cisplatin-resistant patients’ tissue specimens (Figure 1A and B). Pearson’s correlation coefficient curve analysis showed a significant correlation between NEAT1_2 and SFPQ expression levels in all obtained tissue specimens (Figure 1C), while Kaplan–Meier curve analysis of SFPQ expression levels over liver cancer patients’ survival using TCGA public data suggested that high SFPQ expression correlates with unfavorable prognosis. The result was statistically significant, P=0.0003 (Figure 1D). These data preliminarily confirmed the relevance of NEAT1_2 and SFPQ to liver cancer development and cisplatin resistance.
NEAT1 promotes the proliferation of liver cancer cells in vitroNEAT1_2 expression in six different liver cancer cell lines was first evaluated by RT-qPCR, the result of which showed that QGY-7703 had the highest NEAT1_2 expression level, while HUH-7 showed the lowest among the six liver cancer cell lines (Figure 2A). Therefore, QGY-7703 cells were chosen for NEAT1 knock-out and HUH-7 for NEAT1 knock-in before cell viability, proliferation, apoptosis, and colony formation of these two cell lines with different NEAT1_2 expression levels were evaluated. Our data showed that NEAT1 knock-out significantly decreased cell viability and cell proliferation rate of QGY-7703 cells, while NEAT1 knock-in in HUH-7 cells oppositely influenced its cell viability and proliferation rate (Figure 2B–D). NEAT1 knock-out also significantly increased cell apoptosis rate and decreased colony formation ability of QGY-7703 cells, but NEAT1_2 knock-in displayed no significant impact on that of HUH-7 cells (Figure 2E–H). These data suggested that NEAT1 might promote the proliferation of liver cancer cells and might be involved in their self-maintenance in vitro.
NEAT1_2 mediates cisplatin resistance in liver cancer cells partially through SFPQBecause the enzymatic activity of any isoform of NEAT1 has not been reported, we hypothesize that NEAT1_2 does not mediate cisplatin resistance alone but interacts with other mediators. SFPQ or other paraspeckle proteins inter-acting with NEAT1_2 have already been demonstrated to
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facilitate DNA damage repair. We therefore investigated the cisplatin resistance of QGY-7703 and HUH-7 liver cancer cells with different NEAT1_2 or SFPQ expression levels. NEAT1_2 knock-out increased vulnerability of QGY-7703 cells to cisplatin cytotoxicity, which was synergized by SFPQ knockdown, while NEAT1_2 overexpression by knock-in enhanced cisplatin resistance of HUH-7 cells, which was attenuated by SFPQ knockdown (Figure 3). Knocking out NEAT1_2 and knocking down SFPQ simultaneously promotes apoptosis in QGY-7703 liver cancer cells; over-expression of NEAT1_2 and knockdown SFPQ inhibits apoptosis in HUH-7 liver cancer cells (Figure 3A and B). Inhibition of NEAT1_2 and SFPQ expression can inhibit cell
colony formation in QGY-7703 liver cancer cells; NEAT1_2 knock-in and SFPQ knockdown can promote cell colony formation in HUH-7 liver cancer cells (Figure 3C and D). These data suggest that NEAT1_2 is essential for cisplatin resistance of liver cancer cells in vitro, and SFPQ is possibly part of the NEAT1_2 mediated cisplatin resistance mecha-nism. Our data further showed that NEAT1_2 knock-out or knock-in did not significantly change SFPQ transcriptional or translational levels in QGY-7703 or HUH-7 cells, revealed by RT-qPCR (Figure 4C) and Western blotting (Figure 4A and B), suggesting that NEAT1 facilitates cisplatin resistance in liver cancer cells but not by directly increasing increasing SFPQ expression.
Figure 1 NEAT1_2 or SFPQ expression level change correlates with liver cancer development and cisplatin resistance.Notes: (A and B) Comparison of transcription levels of NEAT1_2 and SFPQ in different tissue specimens; data presented as fold change comparing to naïve-adjacent group DIWHU�QRUPDOL]DWLRQ�WR�*$3'+��RQH�ZD�$129$�ZDV�HPSORHG�IRU�VWDWLVWLFDO�VLJQLÀFDQFH�WHVWV�EHWZHHQ�QDwYH�DQG�FLVSODWLQ�UHVLVWDQFH�JURXSV���C) Correlation of NEAT1_2 and SFPQ transcription levels among all obtained tissue specimens. (D) Kaplan–Meier plot of survival of liver cancer patients with different SFPQ transcription levels. Naïve UHIHUV�WR�SDWLHQWV�GLDJQRVHG�RQ�WKH�ÀUVW�YLVLW��FLVSODWLQ�UHVLVWDQFH�UHIHUV�WR�SDWLHQWV�ZLWK�FLVSODWLQ�UHVLVWDQW�SULPDU�OLYHU�FDQFHU��P,������P,0.01.Abbreviations: Ca, cancerous (liver cancer tissue specimens); Ad, adjacent (adjacent non-cancerous tissue specimens); NS, normal saline.
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Figure 2 NEAT1_2 facilitates liver cancer cell activity in vitro.Notes: (A) NEAT1_2 expression levels in six different liver cancer cell lines, revealed by RT-qPCR. (B) Cell viability of QGY-7703 or HUH-7 cells with different NEAT1 JHQH�PRGLÀFDWLRQV���C and D) Representation and statistics of cell proliferation in different cell groups. (E and F) Representation and statistics of cell apoptosis in different cell groups. (G and H��5HSUHVHQWDWLRQ�DQG�VWDWLVWLFV�RI�FRORQ�IRUPDWLRQ�LQ�GLIIHUHQW�FHOO�JURXSV��P,������P,0.001.Abbreviations: Ctl, knock-out or knock-in control (wild type); KO, NEAT1 knock-out; KI, NEAT1 knock-in.
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Figure 3 NEAT1_2 or SFPQ expression is essential for cisplatin resistance in liver cancer cells in vitro.Notes: (A and B��5HSUHVHQWDWLRQ�DQG�VWDWLVWLFV�RI�FHOO�DSRSWRVLV�LQ�4*<������RU�+8+���FHOOV�ZLWK�GLIIHUHQW�1($7��JHQH�PRGLÀFDWLRQV�ZLWK�WKH�SUHVHQFH�RI������µg/mL cisplatin in culture media. (C and D) Representation and statistics of colony formation in different cell groups with same cisplatin treatment as in (A or B���P,0.05.Abbreviations:�&WO��NQRFN�RXW�RU�NQRFN�LQ�FRQWURO��ZLOG�WSH���.2��1($7��NQRFN�RXW��.,��1($7��NQRFN�LQ��1&��QRQ�VSHFLÀF�FRQWURO��VL6)34��6)34�NQRFN�GRZQ�E�VK51$�targeting.
DiscussionChemo-resistance is a major obstacle in clinical liver cancer management. Platinum-based drugs are commonly used in standard chemotherapy, and decreasing the development of platinum drug resistance may significantly improve thera-peutic outcome and patients’ survival from liver cancer. In the present research, our data clearly demonstrated that NEAT1_2 is involved in liver cancer cell chemo-resistance in vitro, possibly in part through interacting with SFPQ. We speculate that NEAT1_2 interacts with SFPQ because Imamura et al found that NEAT1-dependent SFPQ relocation
from promoter region to paraspeckle mediates IL8 expression upon immune stimuli.25
The 3.7-kb NEAT1_1 and 23-kb NEAT1_2, previously named MENε and MENβ, are the two major isoforms of non-coding RNA NEAT1. Their transcriptions are governed by the same promoter but different post-transcriptional 3′-end processing.26,27 It has been well demonstrated that NEAT1_2 but not NEAT1_1 is fundamental for the assem-bly and maintenance of paraspeckle, a subnuclear structure not essential for normal cell growth or mammalian devel-opment but formed in response to DNA damage and that
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NEAT1_2–SFPQ mediates liver cancer cisplatin resistance
paraspeckle promotes oncogenesis, tumor progression and chemo-resistance.18,19,24–28 In the present research, we first investigated NEAT1_1 and NEAT1_2 expression levels in different tissue specimens obtained from liver cancer patients that were diagnosed on the first visit to our department or bur-dened with cisplatin-resistant liver cancer. Our data clearly demonstrated that NEAT1_2 but not NEAT1_1 is the domi-nant NEAT1 isoform for oncogenesis and cisplatin resistance of liver cancer, which was further confirmed by the gain-of-function and loss-of-function assay in vitro. Targeting
NEAT1_2 but not NEAT1_1 on its specific 3′ region by shRNA achieved similar but more transient results (data not shown) compared to the Cas9/sgRNA-based genome editing method we used for the present research.
Cancer cells are known for their genome instability and high mutagenesis rate, which is probably due to the continu-ous cellular stress caused by altered cell proliferation and metabolism machineries, and a strengthened genome repair mechanism is therefore required for preventing cell death caused by the accumulation of DNA damage. Targeting the DNA damage repair pathway has been proposed as a promising strategy against chemo-resistance of different types of cancers.29–31 SFPQ, NONO, and PSPC1 are three characteristic paraspeckle proteins that have been proposed to facilitate DNA damage repair and the consequential chemo- or radio-resistance in cancer cells via seemingly dif-ferent mechanisms.22–24,32,33 In the present research, our data suggested that NEAT1_2 and SFPQ synergistically promote cisplatin resistance in liver cancer cells in vitro, but change in NEAT1 expression did not affect SFPQ mRNA transcription or protein translation. We therefore speculated that NEAT1_2 supports cisplatin resistance in liver cancer cells possibly by functioning as a scaffold for adaptor and effector paraspeckle proteins required for DNA damage repair.
Paraspeckle is a complex protein aggregate. Naganuma et al identified 36 novel paraspeckle proteins, many of which carry RNA binding motifs.26 Paraspeckle has been linked to tumor progression in different aspects by several different studies. While paraspeckle is seemingly unnecessary for homeostasis, the molecular mechanisms of its cancer promoting role remain unresolved. NEAT1_2 is for now the only RNA molecule identified in paraspeckle and is vital for its integrity; thus, targeting this lncRNA may be a possible therapeutic strategy for tumor management with low collateral damage, especially for advanced tumors with metastasis or chemo-resistance.
AcknowledgmentThis research was funded by the 2016 Henan Science and Technology Innovation Outstanding Talent Project (No 164200510010).
DisclosureThe authors report no conflicts of interest in this work.
References1. Chen W, Zheng R, Zhang S, et al. Cancer incidence and mortality in
China, 2013. Cancer Lett. 2017;401:63–71.
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original article © The American Society of Gene & Cell Therapy
Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary signifi-cantly between patients, this approach can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor anti-host reactivity. To address these limitations, we assessed the ability of three different TCR-!-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the high-est disruption efficiency combined with low levels of tox-icity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cel-lular substrates for next-generation immunotherapies.Received 24 July 2015; accepted 18 October 2015; advance online publication 5 January 2016. doi:10.1038/mt.2015.197
INTRODUCTIONT-cell–based immunotherapies utilizing chimeric antigen recep-tor T-cells (CAR T-cells) hold tremendous potential for the treat-ment of malignancies and have shown encouraging activity in early clinical trials.1,2 However, CAR approaches have so far been implemented using autologous patient T-cells, rendering them cumbersome to generate for widespread or urgent use, and poten-tially leading to variable clinical outcomes due to di!erential func-tional properties of each patient’s starting T-cell populations.
Potential approaches to address the variability of autologous approaches include the use of allogeneic T-cells from healthy donors whose functional properties can be carefully de"ned prior to administration to a patient. A drawback of this approach is that the endogenous T-cell receptor (TCR) present on therapeutic T#cells may direct those cells to produce o!-tumor reactivity in the form of gra$ versus host disease.
As a solution to TCR-driven host tissue reactivity, gene-editing nucleases have been employed in order to disrupt components of the TCR.3–6 %e TCR ! chain (TCRa) is encoded by a single TRAC gene and pairs with the TCR " chain encoded by two TCRB genes. As the TCR a/b dimer is essential for a fully functioning TCR complex, disruption of TCRa function has proven the sim-plest approach to elimination of TCR expression and undesired TCR-driven o!-tumor recognition.
Four major classes of gene-editing proteins exist that share a common mode of action in binding a user-de"ned sequence of DNA and mediating a double-stranded DNA break (DSB). Zinc "nger nucleases (ZFN) are heterodimeric arrays that colocalize at a target DNA site. %ey are comprised of individual "nger sub-units that bind DNA and are tethered to the FokI nuclease domain that cleaves DNA. Transcription activator-like e!ector nucleases (TALEN) are comprised of repeating units that bind DNA by vir-tue of a hypervariable two amino acid sequence (repeat variable diresidue) that governs DNA base recognition.7 Similar to ZFNS, TALENs function as dimeric proteins that are fused to the FokI endonuclease domain for DSB generation. Meganucleases (MN) are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site.8,9 %e clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas9 nuclease platform comprised a small guide RNA (gRNA) transcript that
The first three and the last three authors contributed equally to this work.Correspondence: Mark J Osborn, Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, 420 Delaware Street MMC 366, Minneapolis, Minnesota 55455, USA. E-mail: [email protected]
Evaluation of TCR Gene Editing Achieved by TALENs, CRISPR/Cas9, and megaTAL NucleasesMark J Osborn1,2,3, Beau R Webber1, Friederike Knipping4,5, Cara-lin Lonetree1, Nicole Tennis1, Anthony P DeFeo1, Amber N McElroy1, Colby G Starker2,6, Catherine Lee1, Sarah Merkel1, Troy C Lund1, Karen S Kelly-Spratt6, Michael C Jensen6,7, Daniel F Voytas2,8, Christof von Kalle4,5, Manfred Schmidt4,5, Richard Gabriel4,5, Keli L Hippen1, Jeffrey S Miller9, Andrew M Scharenberg10, Jakub Tolar1,3 and Bruce R Blazar1
1Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA; 2Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA; 3Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA; 4 German Cancer Research Center (DKFZ), Heidelberg, Germany; 5Department of Translational Oncology, National Center for Tumor Diseases, Heidelberg, Germany; 6Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, Seattle, Washington, USA; 7Department of Pediatrics, University of Washington, Seattle, Washington, USA; 8Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA; 9Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA; 10Seattle Children’s Research Institute, and University of Washington School of Medicine, Seattle, Washington, USA
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contacts a target DNA sequence via Watson–Crick base pairing and the Cas9 nuclease that cleaves the DNA.10,11
All but CRISPR/Cas9 has been employed for disruption of the TCR complex by targeting either TRAC or TRBC of the TCR. ZFNs delivered as mRNA resulted in gene disruption rates of 27–37% for TRAC and 4–15% for TRBC.3 Delivering this same pair in either integrase-de"cient lentiviral (IDLV) or adenoviral delivery vehicles resulted in TRAC disruption rates of 10% with IDLV and 50% with adenovirus, and ~5% with IDLV and ~40% with adeno-virus for TRBC.4 TALEN mRNA delivery, at maximal rates, has resulted in ~60% TRAC and ~40% TRBC gene disruption.5,12 We (A.M.S.) have previously described a fusion protein of a mega-nuclease to TAL repeats (termed a megaTAL (MT)) and achieved editing rates for TRAC of >60% using a "rst-generation enzyme codelivered with the Trex2 gene product, an exonuclease that increases gene disruption rates.13
While each of these studies has contributed to the "eld, no evaluation has been performed that has focused on relative suit-ability of di!erent nuclease reagents for implementation of TCR disruption in the context of a scalable translatable manufacturing process. Critical components of such an evaluation are reagent optimization, interrogation of o!-target (OT) e!ects, and scal-able manufacturing of gene-edited cell populations. Here, we have evaluated a TCRa-targeted TALEN, the previously described TCRa MT, and TCRa-targeted CRISPR/Cas9 nuclease reagents for editing of the highly clinically relevant TRAC gene target. We observed varying degrees of gene disruption among reagents gen-erated from the three platforms, with a paucity of OT e!ects. %e approach was further validated by generating TCR-null cells that expressed a CD19 CAR construct and a translationally compat-ible manufacturing process to demonstrate the capacity to gener-ate and expand large numbers of engineered cells. %is process is therefore a "rst step toward second-generation T-cells amenable to introduction of potency enhancements and/or as universal donor cells following introduction of CAR and TCR candidates being pursued for translational use.
RESULTSGene-editing platform targeting, architecture, and delivery formatWe generated nucleases from three di!erent platforms each tar-geted to overlapping sequences within exon 1 of the TRAC gene (Figure! 1a and Supplementary Figure S1). %e MT site was nearly identical to the Streptococcus pyogenes (Sp) CRISPR/Cas9 (located on the opposite strand), and the TALEN site covered the same genomic locus (Figure!1a and Supplementary Figure S1) as the previously reported ZFN.3
%e hybrid MT protein contains the I-OnuI LAGLIDADG homing endonuclease architecture that maintains its “central 4” bp recognition domain8,9 but has been repurposed for TRAC gene binding and is fused to 11 TAL repeat regions (Figure! 1b).6 %e TALEN candidate target site contained a 5# T nucleotide and 22 and 18 bp contacting repeat variable diresidues, respectively, with an optimized architecture containing a N-terminal deletion of 152 residues and 63 wild-type TAL sequences at the C-terminus (Figure! 1c).14,15 %e Sp CRISPR/Cas9 platform employs a guide RNA that contacts a target locus possessing a GN20GG sequence
motif that serves to recruit the Cas9 protein to the target site where it induces a DSB (Figure!1d).10,11,16
%e TALENs were assembled using the Golden Gate method-ology into the RCIscript Goldy TALEN backbone that allows for in vitro mRNA production using T3 polymerase.15 Cas9 can be delivered as a protein or, like the MT, can be in vitro transcribed using a T7 promoter. We also tested the gRNA as DNA (plasmid or linear DNA expression fragment) or RNA, for their e&ciency on gene editing (Figure! 1e). %ese RNA species were either tran-scribed in vitro locally or acquired from a commercial vendor as an unmodi"ed RNA or one whose "rst and last three bases contained a 2#-O-methyl (2#-OMe) phosphorothioate modi"cation, respectively.
Evaluation of nuclease activity in transformed T lymphocytesWe "rst tested the MT candidate for activity in Jurkat cells by electroporating 1 µg of mRNA generated following the standard protocol for T7 RNA polymerase that includes addition of an exogenous polyadenylation signal by the E. coli poly (A) poly-merase enzyme. Flow cytometry was employed to quantify TRAC gene disruption rates using an anti-CD3 antibody that recognizes the intact TCR complex. CD3 disruption rates, as evidenced by the lack of cell surface CD3 expression, were ~80% in MT-treated cells, while GFP-treated cells with rates of gene transfer of 95% showed >95% CD3 (Figure!2a).
%e TALEN RCIscript Goldy backbone7,15 contains 5# and 3# UTR sequences derived from the Xenopus "-globin gene, included to increase translation e&ciency.17 Utilizing the T3 polymerase-based system to generate mRNA resulted in TRAC gene disruption rates of ~30% (Figure! 2a). %e T3-based pro-cedure does not include a polyadenylation step, and we hypoth-esized that including this may increase the stability and expression of TALEN mRNA. When we generated T3 mRNA and added a polyA using reagents from the T7 kit, the knockout rates increased to ~60% (Figure!2a). %ese data show an optimized RNA genera-tion procedure for TALENs using the publically available Goldy backbone plasmid.
Next, we determined the optimal reagents for delivery of the CRISPR/Cas9 material via electroporation. Delivery of a plasmid bearing a U6 promoter driving expression of the gRNA versus a linear PCR product lacking the extraneous plasmid backbone sequences was assessed. Cas9 mRNA with a gRNA expressed from a plasmid resulted in ~90% CD3 disruption rates, while the lin-ear gRNA, similar to TALENs, resulted in 60% disruption rates (Figure! 2b). Because of the potential for ectopic DNA integra-tion using plasmid delivery, we next delivered 1 µg of Cas9 mRNA and 500 ng of gRNA transcript that was produced by in! vitro transcription in our laboratory, resulting in low gene-editing rates (Figure!2b). %ese data indicated that electroporated gRNA transcripts may not reach the nucleus at su&cient rates and/or are degraded by intracellular nucleases prior to complexing with the Cas9 protein.18 Recent advances in long synthetic RNA produc-tion allowed us to obtain high doses of the gRNA as an unmodi-"ed or nuclease-protected species by virtue of 2#-OMe–modi"ed bases.19 %e unmodi"ed and modi"ed gRNAs were employed at a dose of 5 µg using either Cas9 protein or Cas9 mRNA. Use of the nuclease-protected modi"ed gRNA resulted in higher editing
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rates than unmodi"ed guide using both the complexed ribonu-cleoprotein format as well as with all RNA delivery approach (Figure! 2b). A representative 'uorescence-activated cell sorting (FACS) plot of Figure! 2 is shown as Supplementary Figure S2, and collectively, our results de"ne the optimal delivery vehicles for each class of nuclease. MT functioned at high levels with in vitro transcribed, polyadenylated mRNA and TALEN activity could be enhanced with the addition of an exogenous polyadenylation sig-nal. For CRISPR/Cas9, the delivery of the gRNA as a modi"ed nuclease-resistant gRNA species yielded maximum editing rates. Using these data, the nucleases were assessed in primary human T#lymphocytes for TRAC gene modi"cation.
Nuclease activity in primary lymphocytesOur experimental schema for performing gene editing in primary T-cells is shown in Figure!3a and employs an initial cellular acti-vation step followed by electroporation-based gene transfer. We employed the optimized delivery platform for each candidate determined in the Jurkat studies: MT T7 polymerase generated polyadenylated mRNA, TALEN T3 mRNA with a polyA, and
Cas9 mRNA or protein with a modi"ed gRNA. In accordance with previous reports that show enhanced nuclease activity in conjunction with a transient “cold shock” at 30 °C incubation,20 we incubated nuclease-treated cells at 30 °C for 24 hours post gene transfer. %e TALEN showed a signi"cant decrease compared to their pro"le in Jurkat cells; moreover, signi"cant toxicity was asso-ciated with TALEN expression in primary T-cells (Figure! 3b). Due to their maximal activity in Jurkat cells, we also tested the MT and CRISPR/Cas9 reagents over several doses and achieved maximal MT-editing rates of ~75% with 2 µg of mRNA and ~85% with Cas9 mRNA and 5 µg of nuclease-protected modi"ed gRNA (Figure! 3b,c). Collectively, our data show that the highest levels of TRAC gene disruption was accomplished using the MT and CRISPR/Cas9 reagents, and a representative FACS plot for these data is shown in Supplementary Figure S3.
Culture, expansion, phenotyping, and CAR transduction of T-cellsToward achieving our goal of generating a pool of cells lacking the TCR, we developed a manufacturing process for CD3-negative cells
Figure 1 TRAC gene targeting and nuclease architecture. (a) Exon 1 of the TRAC locus with the positions of the nuclease target sites shown in relation to one another. (b) TALE recognition code and MT architecture. TALE repeat variable diresidue:DNA base recognition code is represented by colored bars. The amino acid sequences HD recognize DNA base C, NN binds G, NI interacts with A, and NG binds T. Eleven TALE repeat regions are fused to the meganuclease domain by a peptide linker (blue line), and the hybrid protein is termed a megaTAL. The central four bases common to the parental I-OnuI homing endonuclease from which the MT is derived are shown in blue lettering. (c) The dimeric TALEN proteins contain a deletion of 152 amino acids at the N-terminus and maintenance of 63 TAL amino acids at the C-terminus. The individual repeat variable diresidues each bind a single base of DNA and each half array is joined to a subunit of the FokI heterodimeric nuclease. (d) CRISPR/Cas9 architecture. A chimeric gRNA is shown and in purple is the constant portion of the molecule that interacts extensively with the Cas9 protein and the gene-specific component is shown in black letters. The gRNA contacts a target sequence (yellow boxed, black letters) in the context of a–NGG protospacer adjacent motif. The Cas9 protein contains two domains (HNH and RuvC) each responsible for the cleavage of a single strand of DNA. (e) Expression platforms. MT, Cas9, and TALEN mRNA was generated with either a T3 or T7 RNA polymerase promoter and the ‘+/-” refers to the presence or absence of a polyad-enylation signal added in vitro. gRNA was produced as an RNA transcript (blue line), a circular plasmid with a human U6 polIII promoter, or a linear fragment generated by PCR containing the U6 promoter and full-length guide RNA sequences (black circle/line).
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in which we electroporated T-cells at a low, mid, and high scale with either MT or CRISPR/Cas9 reagents. %e experimental schema is shown in Figure!4a and employs the use of IL2, IL-7, and IL-15 and 9 days of culture followed by negative selection using the col-umn free EasySep system that resulted in a purity of 85% that was improved to 99% by repeating the procedure (Figure!4b). TRAC gene-edited cells were then maintained in IL-7 and IL-15 alone. At
the low scale with an initial input (i.e., electroporation) of 200,000 cells, we reintroduced the TRAC gene mRNA by electroporation at day 15 and then restimulated cells with CD3/CD28 activation beads. Control cells cultured in IL-7 and IL-15 cytokines alone maintained a steady state with little proliferation (Figure! 4c, no TRAC no stim). Addition of the CD3/CD28 beads to cells that did not receive TRAC mRNA resulted in a slight proliferative increase,
Figure 2 Nuclease comparison in Jurkat T-ALL cell line. Nucleic acids were delivered by electroporation into Jurkats and 7 days later, the amount of CD3 loss from the cell surface was determined by flow cytometry. (a) The first lane is the GFP transfection control with 95+% CD3 expression levels. Lane two shows the MT disruption rates. Lanes three and four are TALEN optimization conditions. TALEN mRNA was generated from a T3 polymerase promoter without (T3 alone) or with (T3 + pA) a exogenous polyadenylation signal. (b) Cas9 mRNA and protein with vary-ing platforms of gRNA-editing rates. The first lane is GFP followed by a linear DNA fragment encoding the gRNA (Cas9, linear) or circularized gRNA plasmid (Cas9, plasmid) borne expression systems. Following that are a gRNA transcript (Cas9, RNA) produced locally by in vitro transcription and commercially synthesized gRNAs that are unmodified or modified with 2#O-Methyl (2#-OMe) bases that were delivered at doses of 5 or 10 µg by either complexing with Cas9 protein (RNP) or with Cas9 mRNA. Statistical comparisons were done using the Student’s t-test. *,***, and **** represent P values (Student’s t-test) of <0.05, <0.001, and <0.0001, respectively. Data are from four independent experiments.
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Figure 3 Nuclease activity in primary T lymphocytes. (a) Experimental schema. T-cells were isolated from peripheral blood, cultured at a 3:1 CD3/CD28 bead:cell ratio followed by bead removal and electroporation with the indicated dose of nuclease. Cells were cultured transiently for 24 hours at 30 °C. At day 7, gene knockout efficiencies and cellular viability were assessed. (b) CD3 disruption rates using TALEN mRNA. (c) MT mRNA doses of 1, 2, 4, or 8 µg. (d) Cas9 RNP or mRNA with nuclease protected gRNA at 5 or 10 µg. Experiments were done using at least three unrelated donors in quadruplicate. Average CD3 disruption rate with SEM are shown. RNP, ribonucleoprotein. 2#OMe, 2# O-methly RNA. Dashed lines indicate the demarcation of CD3 disruption rates on the left portion of the graph and the cellular viability on the right side.
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most likely due to CD28 stimulation5 or activation of the <1% of CD3+ cells that remain a$er negative depletion (Figure!4b,c, no TRAC+stim). In contrast, transient recapitulation of the CD3 complex by TRAC mRNA electroporation and stimulation with CD3/28 activation beads allowed for a further "vefold expansion (Figure! 4c, TRAC+stim), thus providing the rapid and e&cient generation of TCR-de"cient T-cells from a small starting popula-tion. Because reactivation of the cells may impact their pheno-type, we pursued higher order scaling. %e CRISPR/Cas9 system required 5 µg of modi"ed gRNA and is a dose that may not be conducive for large-scale application, and therefore, we used an input cell number of 500,000 cells. Nuclease treatment, negative selection, and culture of CD3-null cells in homeostatic cytokines allowed for the recovery of 10 times the input number at day 15
of the procedure (Figure!4d). At a high scale with a 2 ( 106 initial cell number using the MT nuclease, we obtained ~108 TRAC-null cells at day 15 (Figure!4e), and we utilized these cells for a com-prehensive 'ow cytometry–based phenotyping panel (Figure!5). %e majority of the cells were CD4+ (Figure!5a) that, importantly, retained e!ector function (Figure! 5b) (e.g., secretion of IL-2, IFNg, GzmA, GzmB). Furthermore, the in vitro culture period needed for TRAC ablation did not a!ect di!erentiative state, as these cells did not demonstrate a senescent (e.g., loss of CD27, CD28, or CCR7; expression of CD57 or KLRG1) or exhausted (expression of PD-1, Tim-3, or CTLA4) phenotype (Figure!5c).21 To validate that the culture conditions and TCR disruption would allow for CAR transduction and antigen-speci"c killing, we used the schema shown in Figure! 6a in which CD19-CAR lentiviral
Figure 4 Expansion and scaling of CD3-negative cells. (a) Experimental schema. T-cells were harvested, activated, electroporated at 48 hours, and transiently cold shocked. During the first 9 days, the cells were grown in the presence of IL-2, IL-7, and IL-15. Following CD3 cell depletion, the cells were maintained in IL-7 and IL-15 until day 15 when they were enumerated and, if indicated, electroporated with TRAC mRNA and restimulated with CD3/CD28 beads in the presence of IL-2. (b) CD3-negative selection. Post-nuclease treated cells were depleted of CD3-positive cells by comple-tion of one (left) or two (right) treatments with the EasySep procedure. (c) Reintroduction of TRAC mRNA. At day 0, 200,000 cells were treated with MT, and at day 15 post sorted, CD3-negative cells were cultured in IL-7 and IL-15 alone (labeled no TRAC no stim) or with a 3:1 CD3/CD28 bead: cell ratio (labeled no TRAC + stim). A third group received 1 µg of TRAC mRNA via electroporation followed by CD3/CD28 bead stimulation (labeled + TRAC + stim). Cell counts were performed at 2, 4, 6, and 8 days post gene transfer. (d) 500,000 cells were treated with 1 µg of Cas9 mRNA and 5 µg of modified gRNA or (e) 2 ! 106 cells were treated with 10 µg of MT. Cells were grown in bulk to day 9 when they were enumerated. Negative depletion was performed, the CD3-null cells were replated in media with IL-7 and IL-15, and cultured to day 15 when they were counted. Experiments are from three donors and are the total from three experiments with four experimental replicates with averages and SEM shown. Arrow indicates CD3 depletion step with subsequent plating of CD3-null cells.
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transduction is performed followed by gene editing. CD3 nega-tive CD4+ or CD8+ CAR transduced cells showed the ability to form lytic granules in response to K562 cells expressing the CD19 antigen (Figure!6b). %ese data demonstrate that our engineering process does not drive the cells toward exhaustion and the cells maintain functionality when expressing an antigen-speci"c CAR.
Unbiased genome level off-target assessmentHaving optimized the conditions to achieve very high levels of on-target genome editing in primary human T-cells, we next turned to the potential for o!-target activity at other sites across the genome. To achieve such an assessment in an unbiased manner at the genome level, we utilized the IDLV gene trapping, linear ampli-"cation–mediated PCR (LAM-PCR), and nonrestrictive LAM-PCR (nrLAM-PCR) methodology22,23 in Jurkat cells (Figure! 7a). Insertion of the IDLV vector into the genome can occur in the pres-ence of DSBs, permanently marking the locus and enabling mapping by LAM and nrLAM PCR (Figure!7a).22,24 DNA breaks can occur naturally in the absence of engineered nucleases due to normal cel-lular physiology25 or genomic fragile spots,26 and these non–nucle-ase-associated events, in the parlance of the assay nomenclature, are termed integration sites (IS).22 IDLVs delivered in tandem with a nuclease results in multiple insertions at either on-target or OT sites and are termed clustered integration sites (CLIS).22 To con-"rm on-target capture, we screened the Jurkat cells by PCR using an LTR and TRAC-speci"c PCR primer set that showed IDLV presence at the TRAC locus only in cells treated with a nuclease (Figure!7b). Subsequent LAM and nrLAM PCR with MiSeq deep sequencing were performed, and at least 178,000 sequence reads were analyzed for each treatment group (Tables! 1 and 2). %ere were 1,491 IS identi"ed for IDLV only treated cells and, impor-tantly, these did not cluster at loci with sequence homology to the
nuclease candidates (Tables!1 and 2). At the TRAC locus in nucle-ase-treated cells, we mapped 36, 15, and 31 CLIS for CRISPR/Cas9, TALEN, and MT, respectively (Table s 1 and 2). Surprisingly, we were not able to detect OT CLIS in the TALEN or CRISPR/Cas9-treated cells. In the MT treatment group, using saturating nuclease expression, we identi"ed a number of CLIS that represented 10 putative OT sites, and these were analyzed further. %e 10 OT sites and their proximity to genes are shown as a percentage of the total OT CLIS in Table!2 columns 3 and 4. Importantly, none of the OT CLIS occurred in exons and four occurred within introns of genes (DR1, KAT2B, PDE11A, and HIAT1 (Tables! 1 and# 2)). To ascer-tain whether these CLIS showed evidence of nuclease activity and repair by nonhomologous endjoining (NHEJ), we deep sequenced the TRAC and 10 OT target sites in Jurkat cells to quantify inser-tion/deletion (indel) frequency. %ese data showed that four of the sites had indel frequencies of greater than 1% at a resolution of at least 7,000 sequence reads (Supplementary Table S1). We next assessed whether these OT sites identi"ed with indel frequen-cies >1% and/or those that occurred within introns in Jurkat cells would be an accurate indicator for OT e!ects in primary T-cells. A pure population of CD3-null cells (by virtue of treatment with 4 µg of MT; Figure!4b) were harvested and analyzed by Surveyor analysis that showed OT activity at the KAT2B locus (Figure!8a), some potential cleavage products for a minimal sequence homol-ogy site at GBP5 (Figure!8b), and no OT activity at the other loci (Figure! 8c). To determine whether OT activity in the KAT2B intron disrupted splicing of the gene, we cloned and sequenced the cDNA and observed an intact open-reading frame, indicating that OT activity did not alter KAT2B splicing (Supplementary Figure S4). Overall, these data highlight the ability of the IDLV LAM PCR-based assay in Jurkat cells to identify OT sites relevant to gene editing processes performed in primary T-cells.
Figure 5 T-cell phenotyping. Cells treated in a manner as those from Figure 4c were harvested at day 15 for flow cytometric analysis for (a) subset composition, (b) cytokines, and (c) surface exhaustion markers. The numbers of CD4 and CD8 cells positive for the indicated marker are shown in each column. In parentheses are the SEM from the individual experiments (n = 3 donors).
Phenotype % Gene edited (SEM)
Cytokine CD4 (SEM) CD8 (SEM)
Granzyme A 46.2 (5.4) 67.3 (1.2)
Granzyme B 24.1 (8.6) 74.6 (10.5)
IFNg+ 35.7 (2.2) 50.3 (6.4)
IL-2+ 94.0 (0.8) 98.9 (0.3)
IL-4+ 3.0 (0.5) 0.5 (0.1)
TNFa+ 91.0 (0.6) 75.9 (7.2)
Epitope CD4 (SEM) CD8 (SEM)
4+ 94.2 (93.3) 62L+ 37.3 (6.6) 13.0 (5.4)
8+ 2 (0.2) 127+ 3.3 (1.2) 0.0 (0)
56+16+ 0.4 (0.38) 160+ 42.2 (1.3) 34.2 (1.1)
14+ 0.007 (0) KLRG1+ 0.1 (0) 0.2 (0.1)
40L+ 39.8 (4.4) 6.7 (1.7)
45RO+ 78.4 (1.7) 10.8 (3.8)
CTLA-4+ 5.8 (0.3) 0.1 (0.1)
LAG-3+ 0.6 (0.2) 2.3 (1)
PD-1+ 2.2 (0.6) 0.6 (0.5)
25+ 2.3 (0.9) 1.9 (1.5)
45RA+ 3.1 (0.9) 22.9 (6.6)
CCR7+ 33.4 (2.4) 16.0 (3)
TIGIT+ 24.4 (2.6) 34.8 (4.9)
TIM-3+ 0.3 (0) 0.1 (0.1)
27+ 42.1 (0.6) 28.5 (7.8)
28+ 90.7 (3.4) 50.8 (1.5)
57+ 10.8 (1.9) 4.7 (1.1)
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DISCUSSIONAt present, multiple clinical trials are open for patient enrolment or are in development that employ adoptive T-cell immunothera-pies achieving tumor targeting based on single-chain variable fragment–based antibodies. In this rapidly evolving "eld, the elimination of the TCR by virtue of engineered nuclease-mediated gene disruption would represent a synergistic approach to ampli-fying the antitumor e!ects of T-cells while removing their pro-pensity for initiating o!-tumor reactivity. Here, we focused on developing a process for the generation of TCR-de"cient T-cells through TRAC gene disruption. Within the scope of this study, we tested several nuclease candidates with overlapping target sequences in exon 1 of the TRAC gene (Figure! 1), allowing us to assess and achieve TCRa gene disruption without confounding positional e!ects.
%e TALEN pair we designed overlapped the previously reported ZFN site and when the TALEN dimers were delivered as mRNA gave similar, albeit slightly lower, rates of editing com-pared to ZFNs (Figure!3).3 %e study by Torikai et!al.3 using ZFNs showed TRAC disruption rates of ~30% using 2.5 µg of mRNA,
and we observed ~10% using 1 µg (2.5 times less) of TALEN. We did not pursue higher doses of TALEN as we observed toxicity associated with TALEN expression (Figure! 3b). Our TALEN disruption rates were signi"cantly lower than that reported by Berdien et! al.5 and Poirot et! al.12 who achieved editing rates of >55% using mRNA. Interestingly, for each study, their mRNA generation procedure included the addition of a polyadenylation signal that we also found to be highly bene"cial to increasing TALEN rates of activity in Jurkat cells (Figure!2). Of further inter-est is that the TALEN target sites of the study by Berdien et! al.5 and Poirot et!al.12 and our CRISPR/Cas9 and MT target sites were either directly overlapping or separated by <10 bp (Figure!1 and Supplementary Figure S1), and in each study, targeting this small stretch of the TRAC gene produced the highest rates of gene edit-ing (Figures!2–4).5,12
%e CRISPR/Cas9 system is a highly 'exible and user-friendly system that in our optimization studies showed the highest levels of TRAC gene disruption (Figures! 2 and 3). We observed poor editing rates when employing gRNA transcripts produced by in!vitro transcription in our laboratory. Of note, Agilent analysis
Figure 6 CAR transduction and antitumor properties of gene-modified cells. (a) Experimental schema. T-cells were isolated, activated, and cultured in the presence of IL-2, IL-7, and IL-15. CD19 CAR lentiviral transduction was performed on day 0 with a self inactivating (SIN) lentiviral construct encoding the CD19R single-chain variable fragment with the CD28 transmembrane domain (CD28 tm), the 4-1BB costimulatory domain (41BB), the CD3-zeta costimulatory signaling domain (&), a self-cleaving T2A picornaviral peptide sequence (T2A), and a non–ligand-binding trun-cated epidermal growth factor receptor (tEGFR). CD3-negative depletion was performed on day 9 with culture overnight followed by incubation of T-cells with K562 or CD19 transgenic K562 cells. (b) Antitumor activity of engineered T-cells. Equal numbers of T-cells and K562 transgenic cells expressing human CD19 (CD19 Tg K562) or CD19-null K562 were incubated and analyzed for degranulation. Shown is a representative FACS analysis on cells from two donors for CD107a with gating on CD4 and CD8 subsets.
Day 0 Day 2 Day 9 Day 10
Isolation andCD3/CD28
stimulation andtransduction
T-cells
0.20
CD4+
FS
C
CD8+
CD107a
0
96.9 2.92
0.65 0.095
96.3 2.91
0.34 0.18
69.9 29.5
0.17 0
92.2 7.63
0.62 0.27
89.9 9.25
0.47 0.33
74.1 25.1
5# LTR 3# LTRSIN.PGK.CD19.CD28tm.41BB
.&.T2A.tEGFR
Bead removaland
electroporation
CD3 depletion24 hours at 30 °C
IL-2, IL-7, and IL-15
KT19/K562
T-cells + K562 T-cells + CD19 Tg K562
a
b
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con"rmed full-length gRNA transcripts of high quality using our procedure; however, our yields were routinely <1 )g/)l, and we theorize that this was responsible for our observed poor editing rates. Indeed, using commercially synthesized gRNA at a dose of 5 )g/)l resulted in ~60+% CD3 disruption rates using Cas9 mRNA (Figures!2b and 3d). Complexing of the gRNA with Cas9
protein has proven to be an e!ective delivery strategy as well,27 and we observed e&cient editing rates in Jurkat and primary T-cells using the ribonucleoprotein approach (Figures! 2b and 3d). A recent study has further shown that that gRNAs contain-ing modi"ed RNA bases that protect from nuclease degradation results in higher editing rates for the IL2RG, HBB, and CCR5.19 In agreement with this, we achieved extremely high gene knock-out rates in Jurkat and primary T-cells using nuclease-protected gRNA (Figures! 2–4). A potential limitation of this application, at present, is the high dose required and associated cost and spe-cialized manufacturing process required to generate and modify RNA oligos of *100 bp in length. As a solution to this, we devel-oped a strategy whereby low or midrange numbers of cells could be employed for CD3 disruption and expansion (Figure! 4). We were able to isolate TCR/CD3-negative cells with >99% purity (Figure!4b)—an extremely high level of purity that we believe is important to achieve for clinical translation, as even a very small number of residual TCR-expressing cells may be capable of caus-ing signi"cant TCR-driven in'ammation and tissue damage. We attempted to expand the puri"ed cells in homeostatic cytokines IL-7 and IL-15 that have been shown to promote maintenance of CD3-null cells (Figure!4c)4,28; however, while the cells maintained a high degree of viability (>95%) in these conditions, they did not robustly expand (Figure!4c). To provide for expansion of the TCR/CD3-de"cient cells to clinically relevant numbers, we rein-troduced the TRAC gene mRNA into the cells via electroporation and stimulated them with CD3/CD28 beads, achieving a consis-tent and rapid "vefold further expansion (Figure!4c). %ese cells were archived and when re-thawed showed only a slight decre-ment in viability (viability# =# 99% pre-cryopreservation versus 85% post-thaw as assessed by trypan blue exclusion). A potential hurdle to this approach is that gene transfer of unactivated T-cells can be low (unpublished observations), and/or reexpansion may alter the cellular phenotype. As such, we utilized CRISPR/Cas9
Figure 7 Off-target genome mapping. (a) Experimental schema for IDLV gene trapping. Nuclease mRNA for MT and TALEN and Cas9 mRNA and gRNA plasmid were introduced into Jurkats followed by transduction with an IDLV expressing GFP and puromycin. The IDLV is integrated into loci where a DNA break has occurred. LAM and nRLAM PCR experimental schema. LTR priming results in linear fragments that are converted to double-stranded DNA products that are barcoded, deep sequenced, and interrogated against the genome for off-target sites. (b) TRAC IDLV gene trapping confirmation at on-target TRAC locus. A PCR using LTR- and TRAC-specific primers (red and blue arrows shown in a) revealed presence of the IDLV cargo at the TRAC locus for all of the nuclease reagents visualized by agarose gel.
5# LTR 3# LTR
LTR-F TRAC -R
CMV.GFP.2A.Puro
IDLVJurkat +
MNTALEN
CRISPR/Cas9
Neg MTTALENCRISPR/Cas9
HiSeq deep sequencing, bioinformaticalsorting, and alignment to reference
human genome
Barcoded LTR: genomic fragmentsLTR primer linear
amplification products
Double-stranded break and viralcargo capture
a b
Table 1 On- and off-target summary: TRAC CLIS
Sample Sequence reads IS On-target CLIS
IDLV 269407 1491 0CRISPR/Cas9 178125 357 36TALEN 299008 437 15MT 316604 1263 31Total number of sequence reads for each sample are shown. LTR:genomic fusion DNA sequences are mapped to identify random integration sites (IS) and clustered integration sites (CLIS) that are the result of on- or off-target events.
Table 2 MT OT sites with number of CLIS shown as a percentage of all recovered sites
MT OT-CLIS Location Distance to gene Gene
62.5% Chr3; intron ~1 kb KAT2B3.9% Chr2; intron ~1 kb PDE11A3.9% Chr1; intron ~5 kb DR12.3% Chr1; intron ~5 kb HIAT14.6% Chr10; intergenic >10 kb KIAA1217/
ARHGAP213.1% Chr5; intergenic >10 kb ADAMTS192.3% Chr1; intergenic >10 kb GBP58.5% Chr1; intergenic >100 kb KCNT27% Chr6; intergenic >100 kb EXOC21.5% Chr4; intergenic >100 kb LEF1The gene and distance from or position within a gene are detailed.
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with an input number of 500,000 cells and were able to obtain a 10-fold higher number of CD3-negative cells (Figure!4d). Using the MT reagent at a nuclease to cell dose of 2 µg of MT:100,000 cells with an input of 2 ( 106 cells, we were able to obtain 108 TRAC-null cells (Figure!4e). %e small, highly active, easily gen-erated MT thus allows for high-level gene disruption at a compar-atively low dose that does not require specialized modi"cations or contracted/specialized generation. Importantly, the cells modi-"ed in this manner retained functionality and did not exhibit a senescent phenotype (Figure!5). Moreover, when transduced with a CAR that recognizes the CD19 surface antigen, the cells were able to form lytic granules at a near-identical rate as reported by Berdien et!al.,5 showing the ability of CAR and nuclease-modi"ed cells to fully function (Figure!6). CAR T-cells, importantly, have shown an ability to expand in the presence of antigen that is pro-vided via arti"cial antigen presenting and retain their ability to lyse cells.3 Our studies complement this approach and represent a manner in which cells can be generated without arti"cial antigen-presenting cells.
In this work, we employed a genome-level methodology to assess nuclease o!-target e!ects for MT, TALEN, and CRISPR/Cas9 head to head for a single, clinically relevant gene (Figure!7). Nuclease speci"city has been assessed by in silico and genomic level assays in relevant cells; however, the computer-based modeling algorithms can be limiting.29,30 A new methodology,
Digenome-seq,31 employs an in vitro, DNA-cleavage methodology to produce Cas9-cleaved identical 5# ends that are sequenced and computationally aligned.31 A potential limitation is that the puri-"ed, cell-free, DNA that is exposed to the nuclease may not faith-fully represent the intracellular architecture in regard to structure and/or chromatin condensation. Our data using IDLV capture and LAM PCR showed the importance of this consideration as the Jurkat IDLV data for MT demonstrated that KAT2B was a bona "de OT site in Jurkat and primary T-cells, while the other Jurkat OT sites were absent or minimally prevalent in primary T-cells (Tables! 1 and 2 and Figure! 8). We hypothesize that this may be directly related to the aneuploid, and likely highly euchromatic, genome of Jurkat cells that would be more permissive to nuclease access to extended portions of the genome. We further hypoth-esize that along with nuclease activity and expression levels at or near saturation (Figure!2), the OT e!ects should be ampli"ed in Jurkat cells making them ideally suited for OT analysis. A caveat to the IDLV approach and other cell-based assays like GUIDE-seq,32 and LAM PCR high-throughput, genome-wide, transloca-tion sequencing (HTGTS)33 is that they all require viable cells for analysis. LAM PCR HTGTS utilizes LAM PCR to identify OT sites by virtue of translocation events that occur between on-tar-get and OT cleavage ends.33 GUIDE-seq uses an oligonucleotide “bait” and single-tail adapter/tag PCR to generate fragments that are deep sequenced and mapped within the genome.32 As such,
Figure 8 Off-target assessment in primary T-cells. Target loci were amplified from 100% CD3-null cells and analyzed by the Surveyor assay for evidence of nuclease activity. (a) KAT2B Surveyor cleavage products and sequence alignment to TRAC. Cleavage products are indicated by arrows and quantitative gel analysis indicating is shown at bottom of gel. (b) GBP5 Surveyor image and genomic sequence in relation to TRAC. (c) PDE11A, DR1, HIAT1, KIAA1217, and EXOC2 Surveyor analysis that did not reveal MT OT cleavage. DR1, downregulator of transcription 1; EXOC2, exocyst complex component 2, TBP-binding (negative cofactor); GBP5, guanylate-binding protein 5; HIAT1, hippocampus abundant transcript 1; KAT2B, K(Lysine) acetyltransferase 2B; KIAA1217, sickle tail; PDE11A, phosphodiesterase 11A. Black arrows indicate cleavage products following Surveyor/CELII enzyme digest. The sequence alignments are in relation to the TAL repeat variable diresidues, the intervening spacer sequence separating the TAL from the meganuclease domain, and the meganuclease domain with the I-OnuI homing endonuclease “central four” ATTC sequence. Mismatches between the TRAC and OT sites are lighter shaded letters.
KAT2BC CD3'
PDE11AC CD3'
DR1C
TAL SPACER MN Central 4
TAL SPACER MN Central 4
CD3'HIAT1
C CD3'KIAA1217
C CD3'EXOC2
C CD3'
GBP5C CD3'
14%
a
b
c
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LAM IDLV, GUIDE-seq, or HTGTS will not be able to identify the full spectrum of OT events if they were to occur within genes that are essential for survival or that generate toxic translocation events. %us, we cannot de"nitively rule out viability-incompat-ible OT events for TALEN and CRISPR/Cas9, particularly with the observed TALEN toxicity in primary T-cells (Figure! 3b). Importantly, the MT and CRISPR/Cas9-treated primary T-cells showed high level TRAC gene disruption with correspondingly high viability and expansion (Figures!3 and 4). %is indicates that the KAT2B OT site that represented 62.5% of the OT CLIS is well tolerated in T-cells. Moreover, the OT site is in the "$h intron of the gene, and the MT-induced indel did not perturb KAT2B splicing (Supplementary Figure S4). Furthermore, we note that OT e!ects with genome-editing reagents are not unprecedented, as evidenced by CCR5 ZFN therapies that have entered the clinic with signi"cant rates of OT cleavage at the CCR2 locus.34 Moreover, our identi"cation of this previously unknown OT site6 is important, as it will guide additional protein engineering of the homing endonuclease cleavage head domain to eliminate OT e!ects in second-generation megaTAL enzymes.22,34 Additionally, by design, our CAR construct also coexpresses a truncated, non–ligand-binding form of the epidermal growth factor gene (tEGFR) (Figure!6) that serves as a safety mechanism to allow for preferen-tial eradication of CAR T-cells by administration of Erbitux. %us, potential adverse events related to OT e!ects, lentiviral transduc-tion, or cellular infusion can be tightly regulated by using a suicide construct.
%is study along with the studies by Poirot et!al.12 and Berdien et! al.5 document the highest reported TRAC gene disruption rates using TALENs. In contrast, our TALEN pair exhibited high toxicity and low activity. Conversely, the MT and CRISPR/Cas9 reagents showed robust TRAC knockout rates. Ostensibly, the dif-ferential targeting sites and their associated accessibility to nucle-ases in regard to epigenetic factors (e.g., chromatin state) will factor in nuclease design and application for the most complete targeting strategy. An important component of this is the ability to assess OT events and to engineer, maintain, and expand the cells in a manner that is clinically viable.
Herein, we document the "rst genome-wide OT screen com-paring candidates from three of the major classes of nucleases. %e MT reagent exhibited OT e!ects that do not appear to disrupt gene expression and the MT’s small size and high rate of editing at low dose makes it an attractive reagent. CRISPR/Cas9 is user friendly and allowed for the generation of a reagent that yielded high TRAC disruption rates and did not exhibit OT activity; how-ever, large doses of synthetic RNA were required for which many laboratories will require commercial acquisition. %ese consider-ations are important, and we extended this to further optimize the engineering process by either expanding gene edited cells by transiently reexpressing TRAC and restimulating the cells or by controlling the initial number of cells as part of a scalable process. Under either condition, we were successful in obtaining adequate numbers of cells such that putative cell or dosing hurdles can be surmounted resulting in a cell population whose dose is within the range of those being used for T-cell therapy.35 Collectively, our
data delineate an approach to determine the critical properties of gene-editing reagents for unique gene targets.
MATERIALS AND METHODSNucleic acid constructs. %e MT was constructed as previously described.6 %e TALEN site was selected using the TAL e!ector Nucleotide Targeter 2.036 and were assembled using the GoldenGate7 methodology and cloned into the RCIscript Goldy backbone.15 CRISPR/Cas9 target site selection and in silico predictive o!-target assessment were accomplished using the CRISPR Design Tool (crispr.mit.edu)37 and constructed using a synthetic G-block gene fragment (IDT DNA Technologies, Coralville, IA). Cas9 plasmid was obtained from Addgene (Cambridge, MA).11
RNA production. %e MT plasmid was linearized with SwaI, TALEN plasmid linearization was accomplished with SacI, and Cas9 was linear-ized with NheI. TRAC (NC_000014.9) cDNA was a linear PCR fragment. One microgram of DNA was used for either T3 (TALEN) or T7 (MT, Cas9, TRAC) promoter generated mRNA in vitro transcription using the mMESSAGE Machine T3 or mMESSAGE Machine T7 Ultra kits (%ermo Fisher Scienti"c, Grand Island, NY). For T3 transcripts that were polyade-nylated, the mMESSAGE Machine T3 procedure was followed by the poly-adenylation step from the mMESSAGE Machine T7 Ultra kit. Guide RNA transcript was performed by amplifying a single-stranded oligonucleotide (target site underlined): (TRAC): AGCGCTCTCGTACAGAGTTGGCAT TATAATACGACTCACTATAGGGGAGAATCAAAATCGGTGAAT GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTT ATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT
using the primers: forward: AGCGCTCTCGTACAGAGTTGG and reverse: AAAAAAAGCACCGACTCGGTGCC with Q5 Hot Start High-Fidelity 2X Master Mix (New England BioLabs, Ipswich, MA). mRNA and gRNA transcripts were DNase treated and then puri"ed using the RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA) with elution in water. Commercially synthesized gRNAs/Cas9 were obtained from TriLink Biotechnologies (San Diego, CA), and the sequences are: unmodi"ed gRNA: 5#- GAG AAU CAA AAU CGG UGA AUG UUU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC UUU U -3. Modi"ed: 5#- 2#OMe(G(ps)A(ps)G(ps)) AAU CAA AAU CGG UGA AUG UUU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC 2#OMe(U(ps)U(ps)U(ps) U -3#. 2#OMe=2#O)-methyl RNA and ps=phosphorothioate. Guide RNAs were reconstituted at 5 )g/)l in Neon Bu!er T.
T-cell isolation and negative selection. Eight microliters of whole blood was obtained by phlebotomy and heparinized with 15 USP of heparin and T-cells were isolated using the RosetteSep Human T-cell enrichment cocktail (StemCell Technologies, Vancouver, BC). Negative selection was performed by subjecting the T-cells to either one or two rounds of deple-tion using the EasySep Human CD3 Positive Selection Kit (StemCell Technologies, Vancouver, British Columbia, Canada).
Cell activation and culture. T-cells were cultured in X-VIVO 20 media (Lonza, Basel, Switzerland) supplemented with 20% AB human serum (%ermo Fisher Scienti"c). For activation, the cells were further supple-mented with recombinant IL-2 (Chiron, Emeryville, CA) at a concentration of 300 IU/ml. Anti-CD3/CD28 Dynabeads (%ermo Fisher Scienti"c) were added at a 3:1 bead:cell ratio, and cells were cultured at 37 °C and 5% CO2 at a concentration of 500,000/ml. Where indicated, recombinant human IL-7 or IL-15 (PeproTech, Rocky Hill, NJ) was included at 5 ng/ml.28
Gene transfer. Forty-eight hours a$er activation, the CD3/CD28 beads were magnetically removed, and the cells were cultured in the absence of beads for 6–12 hours. 200,000 cells were electroporated with the indicated
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amounts of nucleic acid. For the ribonucleoprotein conditions, either 5 or 10 )g of gRNA was incubated with 1 )g of Cas9 protein (%ermo Fisher Scienti"c) in a total volume of 5 )l for 15 minutes at room tem-perature. %e 10 or 100 µl tip of the Neon Transfection System (%ermo Fisher Scienti"c, Grand Island, NY) was used for gene transfer with the following conditions: primary T-cells (in Bu!er T): 1,400 V, 10 ms, 3 pulses. Jurkats (In Bu!er R): 1,325 V, 10 ms, 3 pulses. Cells were plated in 200 µl of antibiotic-free media and cultured at 30 °C for 24 hours. Cells were main-tained at 500,000/ml in media containing antibiotics a$er the initial plat-ing in antibiotic free media.
Surveyor assay. Genomic DNA was isolated using the PureLink Genomic DNA Mini Kit (%ermo Fisher Scienti"c) and ampli"ed with the primers listed below using AccuPrime Taq DNA polymerase, high "delity (%ermo Fisher Scienti"c) as follows: 95 °C#(#2 minutes, and 40 cycles of 95 °C#(#40 seconds, 59 °C# (# 40 seconds, and 68 °C# (# 1 minute. Nine microliters of the PCR product was denatured/renatured with 1( PCR bu!er and then incubated for 20 minutes with the Enhancer and Surveyor enzyme38 (IDT DNA Technologies). Products were then resolved on a 10% TBE PAGE gel (%ermo Fisher Scienti"c, Grand Island, NY) at 200V and stained with ethidium bromide.
Surveyor primers. %e following surveyor primers were used:TRAC F: ATCACGAGCAGCTGGTTTCTTRAC R: CCCGTGTCATTCTCTGGACTKAT2B F: AGCTCTAGGACGATGAGGGAKAT2b R: CCTATCCCACCAGCATCCAAPDE11A F: CAGCATCAGCACTGCACTTTPDE11A R: TGCTGTAGGGCCTGGTTTACHIAT1 F: CTGCTTCCATCTTGCCTCACHIAT1 R: TATCCCCAGCACCCAGTAAGDR1 F: TCCATTGTGTAATGAAGATGATTTGDR1 R: TGCAACAAAATAGGAACACCTTTKIAA1217 F: TGCCTATATTTTCTTCTGTGAGAKIAA1217 R: AGACCTAGAATTGCCAAAACAGBP5 F: TGGTCAAGTGTCGAGTTTGTGBP5 R: ATCCAGTCACCTTCCACCAGEXOC2 F: AGGCCATAGTCACCCAAACAEXOC2 R: TTGGGTTCTTGGTCACGAAG
Flow cytometry. For TCR disruption rate assessment, Jurkats and primary T-cells were cultured for 7 or 9 days post electroporation, respectively, and analyzed for the presence of CD3 by 'ow cytometry. Similarly, phenotypic analysis was performed on day 9 T-cells using the antibodies indicated in Figure! 5. For FACS, cells were washed with phosphate-bu!ered saline and stained at 1:100 dilution with the appropriate antibody (all antibod-ies obtained from eBiosciences, San Diego, CA) for 60 minutes at 4 °C in 'uorescence-activated cell sorting bu!er consisting of phosphate-bu!ered saline#+#1% fetal bovine serum#+#1 mmol/l ethylenediaminetetraacetic acid. Cells were then washed three times in 'uorescence-activated cell sorting bu!er and resuspended in 'uorescence-activated cell sorting bu!er contain-ing Sytox Blue (%ermo Fisher Scienti"c) for dead cell exclusion. Acquisition was performed on a BD FACSCanto (BD Biosciences, San Jose, CA), and data was subsequently analyzed using FlowJo (Tree Star, Ashland, OR).
Off-target analysis, deep sequencing, and KAT2B cloning. Six samples for CRISPR/Cas9 and MT and four for TALEN were generated for OT analysis by electroporating Jurkat cells with 1 )g of MT or TALEN mRNA or 500 ng each of Cas9 mRNA and gRNA plasmid. %e cells were immediately cocultured with a GFP-2A-puromycin IDLV at a multiplicity of infection of 5 with subsequent spinning transduction. Forty-eight hours post gene transfer, 400 ng/ml of puromycin was added, and the cells were expanded for 7 additional days followed by genomic DNA isolation. nrLAM PCR or LAM PCR with MseI or MluCI22,23,39 was performed, and deep sequenc-ing data were generated with the Illumina MiSeq platform (San Diego,
CA). %e high-throughput insertion site analysis pipeline40 was utilized for vector trimming and genome alignment, and IS/CLIS identi"cation was determined.
Total RNA was isolated from MT-treated CD3-negative cells that were sorted to purity using the RNeasy MinElute Cleanup Kit (Qiagen) and reverse transcribed with SuperScript Vilo (%ermo Fisher Scienti"c) and PCR ampli"ed with primers: F:5#-GCCGAGGAGTCTTGTAAATGTAATGG-3# and R:5#- TCACTTGTCAATTAATCCAGCTTCC-3# at 98 °C# (# 2 minutes, 98 °C#(#40 seconds, 58 °C#(#40 seconds, 68 °C#(#2 minutes and 30 seconds for 40 cycles. PCR products were TA cloned into the pCR-4-TOPO Vector and Sanger sequenced with M13 Forward, M13 Reverse, and KAT2B1 internal: 5#-TACCTCGGTACGAAACCACACAGG-3#. Sequence products were aligned to NM_003884.4.
Cytotoxicity assay. VSV-G pseudotyped CD19 CAR-tEGFR lentivirus was produced in 293T cells, and 50 ml of supernatant was concentrated with Lenti-X Concentrator, and the viral pellet resuspended in 2 ml of T-cell culture media. Five hundred microliters of viral supernatant was added to a retronectin-coated plate and spun for 3 hours at 1,200 rpm. 1 ( 106 T-cells were added with CD3/CD28 beads and cultured for 48 hours and then treated with the appropriate nuclease. At day 15, equal numbers (100,000 each) of CD3-negative T-cells and K562 or K562-CD19 transgenic cells were incubated together for 1 hour at 37 °C. Monensin (eBiosciences, San Diego, CA) was then added to the cells followed by an additional 1-hour incubation. %e cells were permeabilzed (A!ymetrix, Santa Clara, CA), stained with anti-CD107a antibody (eBiosciences), and "xed (A!ymetrix) for 'ow cytometric analysis.
Statistics. Student’s t-test was utilized with P#<#0.05 considered signi"cant.
SUPPLEMENTARY MATERIALFigure S1. Individual nuclease target sequences in the TRAC gene.Figure S2. Representative FACS plots for data used in Figure 2.Figure S3. Representative FACS plots for data in primary T-cells used in Figure 3.Figure S4. KAT2B indel and cDNA analysis in primary T-cells.Table S1. Deep sequencing of on and off target loci.
ACKNOWLEDGMENTSThe authors are grateful to Kelsey Vigoren for editorial help with this article. The authors are also grateful to Jordan Jarjour and Alexander Astrakhan for helpful comments and edits. The authors appreci-ate funding support from The Children’s Cancer Research Fund, the Lindahl Family & the Corrigan Family, and the Masonic Cancer Center Cancer Experimental Therapeutics Initiative. B.R.W. is supported by NIH T32- HL007062. J.T. is supported in part by R01 AR063070 and P01 CA065493. M.J.O. is supported by 8UL1TR000114-02. Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1TR000114 (M.J.O.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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