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Cancer Therapy: Preclinical

Untouched GMP-Ready Purified EngineeredImmune Cells to Treat CancerTrudy Straetemans1, Cordula Gr€under1, Sabine Heijhuurs1, Samantha Hol1,Ineke Slaper-Cortenbach2, Halvard B€onig3, Zsolt Sebestyen1, and J€urgen Kuball1

Abstract

Purpose: Engineering T cells with receptors to redirect theimmune system against cancer has most recently been describedas a scientific breakthrough. However, a main challenge remainstheGMP-grade purification of immune cells selectively expressingthe introduced receptor in order to reduce potential side effectsdue to poorly or nonengineered cells.

Experimental Design: In order to test a novel purificationstrategy, we took advantage of a model gdT cell receptor (TCR),naturally interfering with endogenous TCR expression anddesigned the optimal retroviral expression cassette to achievemaximal interference with endogenous TCR chains. Followingretroviral transduction, nonengineered and poorly engineeredimmune cells characterized by a high endogenous abTCR expres-sion were efficiently depleted with GMP-grade anti-abTCR beads.Next, the engineered immune cells were validated for TCR expres-sion, function against a panel of tumor cell lines and primary

tumors and potential allo-reactivity. Engineered immune cellswere further validated in two humanized mouse tumor models.

Results: The untouched enrichment of engineered immunecells translated into highly purified receptor-engineered cells withstrong antitumor reactivity both in vitro and in vivo. Importantly,this approach eliminated residual allo-reactivity of engineeredimmune cells. Our data demonstrate that even with long-termsuboptimal interference with endogenous TCR chains such as inresting cells, allo-reactivity remained absent and tumor controlpreserved.

Conclusions: We present a novel enrichment method forthe production of untouched engineered immune cells, readyto be translated into a GMP-grade method and potentiallyapplicable to all receptor-modified cells even if interferencewith endogenous TCR chains is far from complete. Clin CancerRes; 21(17); 3957–68. �2015 AACR.

IntroductionRising clinical data demonstrate the potency of the adoptive

transfer of T cells to effectively treat cancer, when T cells aregenetically engineered to express tumor-specific receptors (1–4). Although the engineering process of genetically modified Tcells has been substantially improved during the last decades, todate all cellular products usually maintain a fraction of nontrans-duced and poorly transduced immune cells (2, 5). However,nonengineered immune cells have been reported to hamper theactivity of adoptively transferred cells in an autologous situation(6, 7) due to the unwanted transfer of regulatory T cells (8) or bycompeting for homeostatic cytokines (9, 10). In addition, non-engineered or poorly engineered immune cells with high endo-genous abTCR expression have a clear potential to induce graft

versus host disease (GVHD) in the context of allogeneic/third-party applications (11, 12).

To date, efforts to enhance purity of engineered immune cellsmainly focus on positive selection either by expression of anadditional transgene such as truncated CD34 (13), truncatednerve growth factor receptor (14), or direct binding of the intro-duced therapeutic receptor (15, 16). However, these strategies caneither result in reduced expressionof thedesired geneof interest oradditional introduction of potentially immunogenic compo-nents. In addition, the positive selection process results in so-called "touched cells" and can mediate activation-induced celldeath (17, 18) directly after isolation or antibody dependentcellular cytotoxicity shortly after transfer into the recipient andmay hamper long-term persistence of transferred immune cells.An obvious alternative strategy would be the isolation of"untouched engineered immune cells."

Most recently, GMP-grade anti-abTCR beads became availableand are currently used in the context of haploidentical transplan-tation used by others (19) and us (NTR2463 and NTR3079).Using such selection beads in combination with the knockdownof endogenous abTCR genes (20) should theoretically result in apopulation of untouched engineered immune cells with highpurity and substantially reduced "off-target" effects. Despite thefact that various different strategies to knockdown endogenousabTCR expression have been described most recently, includingRNA interference (21, 22), zinc finger nucleases (ZFNs; ref. 20),or TALENS (23), clinical application of these elegant techniques isunfortunately far from translation. Off-target integration effectsand the need for multiple plasmids to be expressed in one T cellare some of the reasons as to why clinical translation has

1Laboratory of Translational Immunology, University Medical CenterUtrecht, Utrecht, the Netherlands. 2Cell Therapy Facility, UniversityMedical Center Utrecht, Utrecht, the Netherlands. 3Institute for Trans-fusion Medicine and Immunohematology, Johann-Wolfgang-GoetheUniversity, Frankfurt, Germany.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

T. Straetemans and C. Gr€under contributed equally to this article.

Corresponding Author: J€urgen Kuball, Department of Hematology, UniversityMedical Center Utrecht, Room number Q05.4.301, Heidelberglaan 100, PO Box85500, 3508 GA Utrecht, the Netherlands. Phone: 31-88-7559030; Fax: 31-88-755-4305; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-2860

�2015 American Association for Cancer Research.

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noticeable obstacles. Alternative simple and cost-effective solu-tions are clearly still needed. Therefore, classical methods suchas the interference with endogenous abTCR chains by intro-ducing physiological strong abTCR competitors for the com-ponents of the CD3 complex (24–26) such as gdTCR chains(27–29), remain valid alternatives in order to substantiallyreduce endogenous abTCR expression. Besides the reductionof endogenous abTCR expression, gdTCRs are promisingimmune receptors to retarget abT cells against cancer asreviewed extensively in refs. 30 and 31. gdT cells are consideredas an innate-like population of immune cells recognizingmolecular stress signals on infected or malignant cells. A subsetof gdT cells express a TCR composed of Vg9 and Vd2 chains thatsense accumulated nonpeptidic pyrophosphate molecules(phosphoantigens), intermediates of a deregulated mevalonatepathway of isoprenoid synthesis, via BTN3A1 (CD277) andreported by us (27–29) and others (32) to display potentcytotoxicity against a broad range of tumor species. Otherintriguing properties of gdTCRs are the absence of formingpotentially self-reactive mixed TCR dimers, their HLA-indepen-dency, absence of immunogenicity, which can either result inreduced therapeutic efficacy or substantial toxicity and avoid-ance of education and long-term tolerance when expressinginnate immune receptors out of the context of an innateimmune cell (30, 31).

In summary, although major progress in the quality andefficacy of clinical engineered T cells has been achieved in thelast decades (1–4), a feasible and cost-effective GMP-compliantsystem to obtain highly purified engineered immune cells is notyet available. By utilizing a novel tumor-specific immunereceptor naturally interfering with endogenous abTCR chains(27–30), an optimized expression system, and clinical gradeanti-abTCR beads, we developed a highly pure engineeredcellular product with improved antitumor activity. The pro-duction process can be readily translated to meet GMP-gradestandards and the resulting cellular medicine can be applied aspart of an immune intervention strategy in a broad populationof cancer patients in autologous, allogeneic, and third-partysituations.

Materials and MethodsCells and cell lines

Daudi (CCL-213) and Phoenix-Ampho (CRL-3213) cellswere obtained from the American Type Culture Collection atthe initiation of the study in 2010 [authentication by shorttandem repeat (STR) profiling/karyotyping/isoenzyme ana-lysis]. OPM2, RPMI8226/S, OPM2-Luciferase (OPM2-Luc), andRPMI8226/S-luc (RPMI-Luc) were kindly provided by AntonMartens in 2012 (University Medical Center Utrecht, Utrecht,the Netherlands) and Daudi-Luciferase (Daudi-Luc) by Gen-mab in 2013 (Utrecht, the Netherlands). The EBV-transformedlymphoblastoid cell lines (EBV-LCL) were a kind gift from TunaMutis in 2010 (University Medical Center Utrecht) and notauthenticated since receipt. All cells were passaged for a max-imum of 2 months, after which new seed stocks were thawedfor experimental use. All cell lines were routinely verified bygrowth rate, morphology, and/or flow cytometry and testednegative for mycoplasma using MycoAlert Mycoplasma Kit.Phoenix-ampho cells were cultured in DMEMþ1%Pen/Strep(Invitrogen) þ10% FCS (Bodinco), all other cell lines inRPMIþ1%Pen/Strepþ10%FCS. Peripheral blood mononuclearcell (PBMCs) were isolated from buffy coats obtained from theSanquin Blood Bank (Amsterdam, the Netherlands) or from theInstitute for Transfusion Medicine and Immunohematology,Frankfurt, Germany. PBMC samples from acute myeloid leu-kemia (AML) patients were a kind gift from Matthias Theobald(Mainz, Germany) and from the University Medical CenterUtrecht Biobank and were collected according to GCP andHelsinki regulations.

TCR gene cassettes in retroviral vectorsThe highly tumor reactive g9d2TCR chain genes were obtained

via combinatorial-gdTCR chain exchange (28). The TCRg chainwas derived from gdT cell clone G115 (33) and TCRd chain fromclone 5 (28), codon optimized (Geneart Life Technologies) andcloned into the retroviral vector pBullet as single TCR chainvectors containing either g-chain-IRES-neomycine or d-chain-IRES-puromycine. In addition, four different transgene cassettescontaining both g and dTCR chains were designed by exchangingtwo different 2A peptide linker sequences, F2A and T2A (34),and the order of TCR chains (d-F2A-g , g-F2A-d, d-T2A-g , g-T2A-d; Fig. 1A). These gdTCR cassettes were cloned into the optimizedretroviral vector pMP71 (kindly provided by Miriam Heemskerk,Leiden University Medical Center, Leiden, the Netherlands) toexpress both TCR chains simultaneously. A nonsense murineabTCR, consisting of the a chain derived from the MDM2/HLA-A2 TCR (35) and the b chain from the p53/HLA-A2 TCR(36), was used as control TCR in both the pBullet and thepMP71 retroviral vector system. Also, truncated nerve growthfactor receptor in pMP71 was used as control in retroviral trans-duction experiments (pMP71:DNGFR; kindly provided by Mir-iam Heemskerk).

Retroviral transduction of T cellsgdTCRs were transduced into abT cells as previously described

(27). In brief, packaging cells (phoenix-ampho) were transfectedusing FugeneHD reagent (Promega) with helper constructs gag-pol (pHIT60), env (pCOLT-GALV) (35) and two retroviral con-structs (pBullet) containing either g-chain-IRES-neomycine ord-chain-IRES-puromycine, or one retroviral construct (pMP71)

Translational Relevance

Although major progress in the quality and efficacy ofclinical engineered T cells for adoptive immune therapiesagainst cancer has been achieved in the last decade, no addi-tional purification step for engineered immune cells is beingapplied due to the lack of suitable tools and strategies. Thisresults in the transfer of also nonengineered and poorlyengineered immune cells into patients, which can substan-tially dampen therapeutic effects and limit additional clinicalapplications such as the transfer of third-party populations. Inthis study, we therefore developed a system to obtain highlypurified gdTCR engineered immune cells that can be readilytranslated into a GMP-compliant production process. Theengineered cells are superior in efficacy, and provide long-term tumor control in twodifferent humanizedmousemodelswithout allo-reactivity. This strategy yields a cellular medicinethat can be part of an immune intervention strategy in a broadcancer patient population.

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Clin Cancer Res; 21(17) September 1, 2015 Clinical Cancer Research3958




containing both gdTCR chains separated by a 2A sequence.Human PBMC were preactivated with aCD3 (30 ng/mL; Ortho-cloneOKT3; Janssen-Cilag) and IL2 (50 IU/mL; Proleukin,Novar-

tis) and transduced twice with viral supernatant within 48 hoursin the presence of 50 IU/mL IL2 and 6 mg/mL polybrene (Sigma-Aldrich). Transduced T cells were expanded by stimulation with

Figure 1.The g-T2A-d TCR transgene cassette improves gdTCR expression and function. A, schematic overview of four different gdTCR transgene cassettes in the retroviralvector pMP71. TCRd chainwas derived from clone 5 (d) and TCRg from cloneG115 (g) (28) and F2A (derived from the foot-and-mouth disease virus) and T2A (derivedfrom the Thosea asigna virus) refer to two different 2A ribosomal skipping sequences. B, following retroviral transduction of abT cells, gdTCR expression wasevaluated by flow cytometry using a pan gdTCR antibody. The percentages gdTCRþ T cells after transduction were calculated as fold increase compared with thepercentage gdTCRþ T cells transduced with the d-F2A-g transgene. Data are presented as mean fold increase of six independent experiments (þSEM) in the leftpanel. Mean fluorescent intensity (MFI) of gdTCR surface expression on gdTCRþ T cells was compared and presented as mean from six independent experiments(þSEM) in the right panel. C, the four different gdTCR-engineered T-cell populations from B were tested for their antitumor function. Tumor cells Daudi (Burkitt'slymphoma) and OPM2 (multiple myeloma) were loaded with 51Cr and incubated with T cells at indicated E:T ratios for 4 to 5 hours. T cells transduced with acombination of two pBullet vectors containing a single MDM2/HLA-A2 TCRa and a single p53/HLA-A2 TCRb chain were used as control T cells (referred to as pB:aMDM2/bp53). gdTCR expression at the time of assay was the following: g-T2A-d: 46%; d-T2A-g : 35%; g-F2A-d: 5%; d-F2A-g : 3.5%. Percentage of specific lysisis shown as mean of triplicates (�SD). D, T cells transduced with the d-T2A-g (20% gdTCRþ) or g-T2A-d (30% gdTCRþ) transgene cassette were incubatedwithDaudi, OPM2,RMI/S8226S tumor cells, or healthydonor–derivedPBMCs in thepresenceof 10mmol/L pamidronate. pB:aMDM2/bp53 transduced cellswere usedas control T cells. After 20 hours of incubation, supernatants were harvested and analyzed for IFNg secretion by ELISA. Data are presented as mean IFNg productionin pg/mL (þSD). Statistical significances were calculated by one-way ANOVA; � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

Purified Engineered Immune Cells

www.aacrjournals.org Clin Cancer Res; 21(17) September 1, 2015 3959




anti-CD3/CD28 Dynabeads (0.5 � 106 beads/106 cells; LifeTechnologies) and IL2 (50 IU/mL) and in case of pBullet retroviralsystem selected with 800 mg/mL Geneticin (Gibco) and 5 mg/mLpuromycin (Sigma-Aldrich). Next, TCR-transduced T cells wereexpanded based on a previously described rapid expansion pro-tocol (REP; ref. 27).

Depletion of nonengineered T cellsabT cells were transduced with pMP71: g-T2A-d and incubated

with a biotin-labeled anti-abTCR antibody (clone BW242/412;Miltenyi Biotec) followed by incubation with an anti-biotinantibody coupled to magnetic beads (anti-biotin MicroBeads;Miltenyi Biotec).Next, the cell suspensionwas appliedonto anLDcolumn and abTCR-positive (abTCRþ) T cells were depleted byMACS cell separation according to the manufacture's protocol(Miltenyi Biotec). After depletion, gdTCRþ T cells were expandedusing T-cell REP.

Flow cytometryAntibodies used for flow cytometry included: pan-gdTCR-PE

(clone IMMU510), pan-abTCR-PE-Cy5 (clone IP26A; both Beck-man Coulter), mouse TCRb-chain-PE (clone H57-597), CD4-FITC (clone RPA-T4; both BD Biosciences), CD8-PerCP-Cy5.5(clone RPA-T8; Biolegend), CD20 (Rituximab; Roche), and Goat-anti-Human-IgG-PE (Jackson ImmunoResearch Laboratories).All samples were analyzed on a FACSCanto II using FACSdivasoftware (BD Biosciences).

Functional T-cell assays51Chromium-release assay for cell-mediated cytotoxicity

was previously described (28). In brief, target cells were labeledovernight with 100 mCu 51Cr and incubated for 4 to 5 hwith transduced T cells in five effector-to-target ratios (E:T)between 30:1 and 0.3:1. Percentage of specific lysis was calcu-lated as follows: (experimental cpm � basal cpm)/(maximalcpm � basal cpm) � 100 with maximal lysis determined in thepresence of 5% triton and basal lysis in the absence of effectorcells.

IFNg ELISPOT was performed using anti-hu IFNg mAb1-D1K(I) and mAb7-B6-1 (II) (Mabtech) following the manufac-turer's recommended procedure. Target and effector cells (E:T 1:3) were incubated for 24 hours in the presence of pami-dronate (10 mmol/L; Calbiochem) where indicated.

IFNg ELISA was performed using ELISA-ready-go! Kit(eBioscience) following manufacturer's instructions. Effector andtarget cells (E:T 1:1)were incubated for 24hours in the presence ofpamidronate when indicated.

TCR surface expression assay was performed with T cells thatwere rested for 21 days and starved of fresh medium and IL2supplement for 6 days. T cells were either stimulated in a 24 wellsplate with anti-CD3/CD28 Dynabeads at a bead-to-cell ratio of1:1 in a final volume of 2 mL medium or with EBV-LCLs in a 96wells plate at an E:T ratio of 1:3 in a final volume of 200 mLmedium supplemented with 30 IU/mL IL2. After a stimulationperiod of 4, 24, and 48 hours at 37�C and 5% CO2, cells werestained with mAbs against the introduced and/or endogenousTCR and analyzed by flow cytometry. TCR cell surface expressionwas calculated as follows: [mean fluorescence intensity (MFI) of Tcells stimulated with beads/MFI of unstimulated T cells] � 100and indicated as % TCR of control.

Animal modelsThe RAG2�/�/gc�/�-BALB/C mice, originally obtained from

AMCAS b.v., were bred and housed in the specific pathogen-free(SPF) breeding unit of the Central Animal Facility of UtrechtUniversity. Experiments were conducted according to Institu-tional Guidelines after acquiring permission from the localEthical Committee and in accordance with current Dutch lawson Animal Experimentation. For the experiments, female micefrom 8 to 12 weeks of age were used. 107 gdTCR or Mock TCR–transduced T cells were intravenously injected together with 0.5� 106 Daudi-Luc or 5 � 106 OPM2-Luc cells. Mice received 0.6� 106 IU of IL2 in IFA subcutaneously on day 1 and every 21days till the end of the experiment. Pamidronate (10 mg/kgbody weight) was applied in the indicated groups at day 1intravenously and every 21 days until the end of the experi-ment. For the OPM2 tumor rechallenge experiment, micetreated with gdTCR T cells that remained tumor free for morethan 120 days were rechallenged with 5 � 106 OPM2-Luc cellswithout prior irradiation. Na€�ve and non-irradiated mice wereused as control for tumor outgrowth. Tumors were visualized invivo by bioluminescent imaging. Mice were anesthetized byisoflurane before they received an intraperitoneal injection(100 mL) of 25 mg/mL Beetle Luciferin (Promega). Biolumi-nescence images were acquired by using a third-generationcooled GaAs intensified charge-coupled device camera, con-trolled by the Photo Vision software and analyzed with M3

Vision software (all from Photon Imager; Biospace Laboratory).

Statistical analysesDifferences were analyzed using indicated statistical tests in

GraphPad Prism (GraphPad Software Inc.).

ResultsOptimal competition and expression of introduced TCR chainswith an individualized transgene cassette

The first goal of the study was to obtain the strongest antitumorreactivity and maximal interference with endogenous abTCRs(24), by no other means than selectively introducing optimally astrong competitor for components of the CD3 complex such as anovel and highly tumor-reactive g9d2TCR (gG11/d5), obtainedby combinatorial-gdTCR-chain exchange (28).General consensusis lacking, regarding the optimal TCR transgene cassette foradoptive cell therapy with TCR-engineered T cells (37–41). There-fore, we designed four different gdTCR transgene cassettes (Fig.1A) to test both the influence of gdTCR-chain orientation asdescribed forabTCR transgene cassettes (37) aswell as a particular2A ribosomal skipping sequence, F2A versus T2A (34). All geneswere codon optimized, cloned into the clinical-grade retroviralvector pMP71 (42), and introduced into abT cells from multipledonors in order to test general applicability. Placing the g chain 50

and the d chain 30 of the F2A sequence (g-F2A-d) increasedtransduction efficiencymore then 2-fold, depicted as fold increaserelative to transduction efficiency of the d-F2A-g cassette andcorresponded with significantly higher TCR expression (Fig. 1B,left). However, the T2A sequence significantly enhanced trans-duction efficiency and MFI for both gdTCR chain orientations(Fig. 1B, right). Flow cytometric plots from a representativeexperiment with the four gdTCR transgene cassettes are shownin Supplementary Fig. S1. To determine whether the increasedgdTCR expression was associated with increased specific cytolytic

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capacity, transductants were cocultured with Daudi (Burkitt'slymphoma) or OPM2 (multiple myeloma) cells (Fig. 1C). T cellstransduced with d-F2A-g did not induce significant lysis oftumor cells and an opposed orientation of g- and d-chain couldonlyminimally increase lysis at the highest effector-to-target (E:T)ratio. In contrast, T cells transduced either with d-T2A-g or g-T2A-defficiently lysed target cells. We used T cells transduced withboth T2A containing transgene cassettes to examine whether thegdTCR chain-orientation influenced tumor-specific release ofeffector cytokines (Fig. 1D). Although lytic activity was not sig-nificantly enhanced, g-T2A-d transductants produced significanthigher amounts of IFNg compared with T cells transduced withd-T2A-g and argued for the use of the g-T2A-d as our clinicalcandidate transgene cassette. Given these results, it is worthwhileto individually optimize transgene cassettes for each particularclinical candidate TCR in order to achieve the most efficientcompetition with endogenous abTCR chains as well as the stron-gest expression and activity of introduced receptors.

Enrichment of untouched engineered T cells using aGMP-gradeantibody

Although retroviral transduction efficiencies have been greatlyincreased over the past decades and clinical abTCR-engineered T-cell products contain up to 60% to 85% TCRþ T cells, theinterpatient variation remains significant (2, 43). To overcomethis problem and obtain a pure gdTCR-engineered T-cell productfor clinical application, we introduce a new procedure for thedepletion of nontransduced or poorly transduced T cells, takingadvantage of the observation that upon introduction of a gdTCR,the endogenous abTCR expression is decreased (refs. 27 and 29;and Supplementary Fig. S1 and Fig. 2A). This procedure is basedon a monoclonal antibody against the abTCR complex (cloneBW242/412; Miltenyi Biotec), currently used to deplete abT cellsfrom stem cell grafts resulting in amean 4 logs abT-cell depletion(NTR2463, NTR3079, and ref. 19). In short, abT cells weretransduced with pMP71:g-T2A-d and incubated with the bio-tin-labeled anti-abTCR antibody followed by incubation withan anti-biotin antibody coupled to magnetic beads for the deple-tion of abTCRþ T cells by MACS cell separation. After depletion,gdTCR T cells were expanded utilizing a previously described T-cell expansion protocol (44). This procedure resulted in nearcomplete depletion of single abTCRþ T cells (from 51% to0.4%) and a dramatic increase in gdTCR single-positive T cells(from 20% to 77%; Fig. 2A). Importantly, the abTCR/gdTCRdouble-positive T cells that remained were characterized by rel-ative low surface expression of the endogenous abTCR. Thisphenotype was stable until day 5 after stimulation, when T cellswere highly activated and proliferative. However, the phenotypechanged at day 9 towards a major population of abTCR/gdTCRdouble-positive T-cells (68%) and a decreased percentage ofgdTCR single-positive cells (25%) when T cells reside in a moreresting phase (Fig. 2A).

The reappearance of endogenousabTCR chains inmore restingcells allowed us to test whether early abTCR T-cell depletion stillprovides a functional advantage when interference with endog-enous TCR chains is far from complete. The functionality of thepurified engineered immune cell product was therefore tested 10days after selection and expansion and compared with a bulkengineered cell product without abTCR depletion. Indeed,abTCR T-cell depletion significantly increased specific lysis ofDaudi cells (P < 0.01; Fig. 2B) as well as IFNg production in

response to three different tumor cell lines (P < 0.001; Fig. 2C).These results demonstrate that abTCR T-cell depletion increasedthe antitumor potential of the engineered cell product.

In order to test whether highly purified engineered immunecells in a resting state with suboptimal expression of introducedgdTCR chains still provide a therapeutic advantage comparedwithprimary gdT cells with optimal gdTCR expression but polyclonalg9d2TCR usage, antitumor activity of primary bulk g9d2T cellsand GMP-grade T cells engineered to express an optimizedg9d2TCR were tested at day 10 against a panel of primaryleukemic cells from AML patients. Treatment of leukemic cellswith pamidronate to block the mevalonate pathway downstreamto induce accumulation of isopentenyl pyrophosphate resulted inIFNg secretion by T cells in response to 9 of 16 AML samples. Infive of eight tested samples, the optimized gdTCR-engineered Tcells produced significantly enhanced levels of IFNg comparedwithprimary polyclonal g9d2T cells isolated fromahealthydonor(Fig. 2D). This result suggests that transfer of optimized engi-neered gdTCR T cells, as part of a clinical immune interventionstrategy, is preferred over a polyclonal gdT cell product. Together,we propose here a depletion procedure using a GMP-gradeantibody for the enrichment of untouched gdTCR engineered Tcells resulting in a highly tumor-reactive clinical cell product evenin the absence of a complete interference with the expression ofendogenous abTCR chains.

Abolished allo-reactivity following enrichment of engineered Tcells

The enrichment procedure that resulted in a pure gdTCR-transduced T-cell population with low or absent expressionof abTCR may reduce the allo-reactive potential of the T-cellproduct in an allogeneic setting. However, this advantagecould theoretically be counterbalanced by the latter upregula-tion of endogenous abTCRs in resting engineered immunecells (Fig. 2A). To further simulate a resting T-cell followingin vivo transfer with substantial reoccurring expression ofendogenous abTCR chains, we pushed the system to a greaterextend by using engineered T cells that lacked stimulus formore than 20 days and were starved of IL2 for 6 days. Mock(DNGFR transduced), gdTCR-engineered, and gdTCR-engi-neered abTCR-depleted T cells were tested against a panel of13 mismatched EBV-LCL cell lines or healthy donor–derivedPBMCs in an IFNg ELISPOT assay (Fig. 3A and B; see Fig. 3C,top, for gdTCR vs, endogenous abTCR expression in rested Tcells). Although Mock T cells produced IFNg in response to 9out of 13 EBV-LCL cell lines, allo-reactivity of gdTCR-engi-neered bulk T cells was greatly reduced (significant reductionfor eight of nine EBV-LCL lines) and more importantly evencompletely abolished in the gdTCR-engineered abTCR-deplet-ed T-cell population. The reduced allo-reactivity of gdTCR-engineered T cells was even more apparent when the differentT-cell populations were tested against a panel of 20 differenthealthy donor–derived PBMCs (Fig. 3B). No allo-reactivity wasdetected in the gdTCR-transduced T-cell populations, but MockT cells produced IFNg in response to 9 out of 10 PBMC donorcombinations. Importantly, although allo-reactivity was abol-ished, both gdTCR-engineered populations maintained theirantitumor reactivity (Fig. 3A and B).

One possible explanation for the reduced allo-reactivity ingdTCR-transduced T cells despite their significantly recoveredendogenous abTCR may be the preferential hit of memory T






Figure 2.Improved antitumor activity of pure gdTCR-engineered T cells after abTCR T-cell depletion. Enrichment of gdTCR engineered T cells by GMP grade depletion ofabTCRþ T cells. A, flow cytometric representation of pMP71:g-T2A-d–transduced abT cells before and directly after abTCR T-cell depletion (abTCRT-cell depleted). Depleted T cells were followed up during T-cell expansion for their gdTCR transgene as well as endogenous abTCR expression using a pan-gdTCR and a pan-abTCR antibody for flow cytometry analysis. Percentages of cells in each quadrant are indicated. B, gdTCR-transduced T cells (bulk, 9%gdTCRþ) and abTCR-depleted (41% gdTCRþ) T cells were incubated with 51Cr loaded Daudi cells at indicated E:T ratios for 4 to 5 hours. pB:aMDM2/bp53-transduced cells were used as control T cells. Percentage of specific lysis is shown as mean of triplicates (�SD). Statistical significances were calculatedby two-way ANOVA; �� , P < 0.01; �� , P < 0.001. C, gdTCR-transduced bulk (6% gdTCRþ) and abTCR-depleted (51% gdTCRþ) T cells were incubated withdifferent tumor target cells as indicated and IFNg secretion was measured by IFNg ELISPOT. pMP71:DNGFR-transduced T cells were used as controlT cells. IFNg spots per 15,000 T cells are shown as mean of triplicates (þSD). T cells did not produce any significant levels of IFNg . Statistical significances werecalculated by two-way ANOVA; � , P < 0.05; �� , P < 0.01; �� , P < 0.001. D, primary AML tumor samples (AML 1-16) were incubated with gdTCR-transducedT cells that were abTCR depleted (>70% gdTCRþ) or with a bulk population of primary gdT cells (only AML 1-9) with or without 10 mmol/L pamidronate(PAM) and IFNg secretion was measured by ELISPOT. IFNg spots per 15,000 T cells are shown as mean of triplicates (þSD). Fifty spots/15,000 cellswere considered as a positive antitumor response and indicated by the black horizontal line.

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cells and not allo-reactive na€�ve T cells in the transduction pro-cedure. However, this explanation could be excluded becauseCD45RA-depleted (memory) and CD45RO depleted (na€�ve) Tcells were transduced with identical transduction efficiencies(data not shown). Meanwhile, reduced surface expression of theendogenous abTCR in gdTCR-transduced T cells may partially

account for reduced allo-reactivity. In unstimulated resting cells,the total amount of abTCR, as indicated by the MFI, in gdTCRengineered cells was reduced for bulk cells by 4% (MFI: 12546)and for abTCR-depleted cells by 14% (MFI: 11334) when com-pared with DNGFR-transduced cells (MFI: 13117; Fig. 3C, top).Finally, a preferential recovery of introduced as compared with

Figure 3.Abolished allo-reactivity but preservedantitumor activity of rested gdTCR-engineered T cells after abTCR T-celldepletion. T cells were retrovirallytransduced with pMP71:g-T2A-d,enriched for gdTCR transduced T cells(abTCR depleted and 65% gdTCRþ) ornot (bulk and 9% gdTCRþ), orpMP71:DNGFR as control and expandedas described. T cells were culturedwithout stimulus for more than 20 daysand starved of IL2 for the last 6 days andconsidered to be resting T cells. A,resting T cells were cocultured withOPM2 tumor cells and a panel ofHLA-mismatched EBV-LCLs for 24hours and IFNg ELISPOT assaywas usedto measure both antitumor activity andallo-reactivity. IFNg spots per 15,000T cells is shown as mean of triplicates(þSD). B, a panel of 20 HLA-mismatched healthy donor–derivedPBMCs were irradiated and cells fromtwo different donors were mixed,indicated by the donor numbers, andused as allo-reactive target cells.Resting T cells were cocultured withDaudi tumor cells and the allo-reactivetarget cells for 24 hours and IFNgELISPOT assay was used to measureboth antitumor activity and allo-reactivity. IFNg spots per 15,000 T cellsis shown as mean of triplicates (þSD).C, T cells described in A and B wereeither left untreated (rested T cells, top)or incubatedwith anti-CD3/CD28 beadsfor 4 days (aCD3/CD28 stimulated Tcells, bottom). T cells were stained witha panabTCR- and pan gdTCR-specificantibody and analyzed by flowcytometry. Percentages of cells in eachquadrant and the MFI of abTCRþ cellsare indicated. Statistical significanceswere calculated by two-way ANOVA;� , P < 0.05; �� , P < 0.01; �� , P < 0.001.






Straetemans et al.





endogenous TCR chains upon antigen encounter may also con-tribute to this effect. Indeed, upon stimulation of resting T cellswith anti-CD3/CD28 beads, the expression of the retrovirallyintroduced gdTCR decreased less profoundly, was restored within24 hours and reached higher levels than before T-cell stimulationwithin 48 hours (Fig. 3C, bottom, and Supplementary Fig. S2A–S2D). This quick restoration and high level of TCR surfaceexpression following T-cell reactivation was not restricted togdTCRs, thus most likely a property of the expression systemused here, because expression kinetics of a retrovirally introducedabTCR were comparable (Supplementary Fig. S2). These resultssuggest that although the endogenous abTCR is reexpressed inresting engineered T cells, reactivation preferentially decreasesendogenous abTCR expression (Fig. 3C, bottom). To formallyaddress this hypothesis in a more physiologically relevantsetting, we cocultured resting gdTCR-engineered abTCR-deplet-ed T cells with an HLA-mismatched EBV-LCL that induced anallo-reactive IFNg response by DNGFR-transduced cells (LCL1from Fig. 3A). Co-culturing these cells resulted in a down-regulated endogenous abTCR expression, which reached aminimal level after 48 hours, whereas the gdTCR expressionremained largely stable over time (Supplementary Fig. S2E).Thus, we confirm that in gdTCR-engineered T cells T-cell reac-tivity, such as allo-reactivity, is prevented and the expression ofretrovirally introduced tumor-specific TCRs is favored andantitumor reactivity maintained. These data question the needof a complete elimination of endogenous abTCR chains inpurified engineered immune cells when expressing strongabTCR competitors with an optimal expression system.

Improved in vivo tumor control by optimized engineered T-cellproduct

Weevaluated the clinical potency of theoptimized gdTCRT-cellproduct in relation to gdTCR-engineered T cells producedwith theextensively used pBullet retroviral transduction and antibioticselection system (45), referred to as pB:gdTCR T cells. Followingtransduction of peripheral blood abT cells and selection withantibiotics (pBullet) or enrichment with abTCR depletion beads(pMP71) and subsequent T-cell expansion, both immune pro-ducts were evaluated. Interestingly, not only the percentagegdTCRþ T cells was higher for the abTCR-depleted gdTCR T-cellproduct, but also the number of gdTCR complexes per cellincreased more than 2-fold compared with pB:gdTCR T cells asmeasured byMFI (Fig. 4A). In addition, lysis of three tested tumor

cell lines was significantly enhanced by abTCR-depleted gdTCR Tcells transduced with the pMP71:g-T2A-d vector cassette as com-pared with pB:gd TCR T cells (Fig. 4B). To test if the superiorantitumor activity of abTCR-depleted gdTCR T cells is reflected invivo, we used a humanized mouse tumor model for adoptivetransfer of gdTCR-engineered T cells. Irradiated RAG2�/�gc�/�

double-knockout mice were injected with Luciferase-positiveDaudi tumor cells and eitherwith gdTCRorMockTCR-engineeredT cells and tumor growth was evaluated by bioluminescenceimaging. Both gdTCR-engineered T-cell products significantlyinhibited tumor growth compared with Mock TCR T cells, butabTCR-depleted gdTCR T cells further delayed tumor outgrowthand significantly increased survival compared with pB:gdTCR Tcells (Fig. 4C).

In a second tumor model of multiple myeloma, the antitumoractivity of the optimized abTCR-depleted gdTCR-engineeredcell product was assessed. Irradiated Rag2�/�gc�/� double-knock-out mice were injected with Luciferase-positive OPM2 cellsand gdTCR or Mock TCR-engineered T cells and tumor growthwas evaluated by bioluminescence imaging. Interestingly, tumorgrowth was completely prevented by clinical-grade gdTCR T cellsin four of seven mice (Supplementary Fig. S3). One hundred andtwenty days after first tumor and T-cell injections, tumor freemice were rechallenged with a second injection of tumor cellswithout prior irradiation and nonirradiated na€�vemice were usedas control for tumor outgrowth. Selectively rechallenged miceremained tumor free, indicating that adoptive transfer of engi-neered immune cells can mediate long-term tumor protection invivo (Fig. 4D). In summary, these data underscore the potency of acost-effective purified engineered immune cell product for clinicalapplication taking advantage of an optimal expression system,strong competitors for endogenous TCR chains such as a gdTCR,and GMP-grade anti-abTCR beads.

DiscussionAdoptive cell therapy using gene-engineered lymphocytes has

moved towards a feasible and effective treatment modality forcancer as demonstrated by exciting clinical results during the lastdecade (1–5). However, large-scale clinical implementation ofthis promising immunotherapy still awaits some critical hurdlesto be taken such as the generation of a purified engineeredimmune product. We provide evidence that the combination ofan optimized and individualized expression system with strong

Figure 4.Improved tumor control of purified gdTCR T-cell product. T cells were retrovirally transduced with pMP71:g-T2A-d, pB:g/d and pB:aMDM2/bp53 as control TCR andunderwent abTCR T-cell depletion in case of pMP71:g-T2A-d or antibiotic selection in case of pBullet vector-engineered T cells prior to expansion. pMP71:g-T2A-drefers to abTCR T cell–depleted engineered cells. A, gdTCR expression was evaluated by flow cytometry and histogram plots illustrate gdTCR expressionandMFI values are indicated. B, T cellswere incubated for 4 to 5 hourswith 51Cr loadedDaudi, OPM2, or RPMI tumor cells at indicated E:T ratios. Percentage of specificlysis is shown as mean of triplicates (�SEM). Statistical significances of pB:g/d versus pMP71:g-T2A-d T cells were calculated by one-way ANOVA; � , P < 0.05;�� , P < 0.01; �� , P < 0.001. C, RAG2�/�gc�/� double knockoutmice were irradiated on day 0 and injected intravenously with 0.5� 106 Daudi-Luc cells and 107 gdTCRengineered or control T cells at day 1. Mice were injected subcutaneously with 6� 105 IU IL-2 in IFA and intravenously with pamidronate (10 mg/kg body weight) atday 1 and every 3 weeks until the end of the experiment. Bioluminescence imaging was used to monitor tumor growth every 7 days. Data represent mean of allmice per group (n ¼ 4–7; �SEM) and is presented in the left panel. Statistical significances were calculated by one-way ANOVA; � , P < 0.05; ��, P < 0.01.Overall survival of treatedmicewasmonitored until the end of the experiment and is presented in the right panel. Statistical significanceswere calculated by log-rank(Mantel–Cox) test; � , P < 0.05; �� , P < 0.01. D, RAG2�/�gc�/� double knockout mice were irradiated on day 0 and injected intravenously with 5� 106 OPM2-Luc cellsand 107 gdTCR-engineered or control T cells at day 1 (see result cancer free survival in Supplementary Fig. S3). Mice that remained tumor free until day120 (four of seven) were rechallenged with 5 � 106 OPM2-Luc cells without prior irradiation and five nonirradiated na€�ve mice were used as control mice. Tumorgrowth was measured as in C and presented in the left panel. Statistical significances were calculated by two-way ANOVA; �� , P < 0.01. Cancer-free survival ofrechallenged mice was monitored and presented in the right panel. Statistical significances were calculated by log-rank (Mantel–Cox) test; �� , P < 0.01.






abTCR competitors and clinical anti-abTCR beads is a highlyefficient system, even if interference with endogenous abTCRexpression is far from complete. Thus, we present to our bestknowledge the first enrichment procedure using a clinical-gradeanti-abTCR mAb for the production of untouched gene-engi-neered immune cells suitable for autologous, allogeneic, or third-party immune intervention platforms, which can be valuable forexpression-receptor formats interfering with endogenous abTCRchains.

Optimal gene expression is crucial when interference withabTCR chains is not mediated via siRNA (21), ZFN (20),or TALENS (23). Therefore, an individualized testing of existingexpression systems was essential when utilizing a naturaloccurring strong competitor for the CD3 complex (27).Although virus-derived 2A "ribosomal skipping sequences" arewidely used to obtain equimolar expression of introducedgenes, there are a handful of different 2A sequences available.A T2A sequence was superior to the F2A and we can unfortu-nately not provide a reasonable explanation for this observa-tion. Our data revealed a preferred position of the TCRd chaindownstream of the 2A cleavage element and this is in line withan optimal abTCR gene cassettes described by Leisegang andcolleagues (37).

Our selection procedure utilizes a GMP-grade clinical bead,which is currently employed in the stem cell transplantation fieldby others (19) and us (NTR2463 and NTR3079) in order todeplete abT cells from the donor. This procedure resulted inremoval of nontransduced and poorly transduced bystander cells,improved gdTCR surface expression levels, and resulted in almost100% gdTCR-engineered cells in the end product, which wastranslated into an increased antitumor function both in vitro andin vivo. Interestingly, when compared with a nonengineeredpolyclonal primary gdT-cell population, selected gdTCR-engi-neered T cells not only recognize an increased number of primaryAML patient samples but also clearly produce higher amounts ofIFNg , in case the tumor cells are recognized. This encouragingfeature of our engineered immune cells is likely due to the choicefor a high avidity gdTCR in contrast to a polyclonal usage ofgdTCRs in a primary gdT-cell population in combination withhigh gdTCR surface expression as a result of the selectionprocedure.

The substantial upregulation of endogenous abTCR chainsin resting engineered immune cells allowed us to thoroughlyinvestigate pitfalls, if interference with endogenous abTCRchains is not complete. Even after 3 weeks, tumor controlremained preserved in engineered immune cells in vitro andin vivo and allo-reactivity could not be observed. This is mostlikely due to (i) a still reduced surface expression of abTCRwhen compared with nonengineered cells and (ii) faster andhigher upregulation of introduced TCR surface expression afterantigen encounter. This observation is in line with previousdata showing that in contrast to the endogenous TCR, intro-duced TCR expression under the control of an optimized viralpromoter is restored, and increased shortly after antigen-spe-cific stimulation of either the endogenous or introduced TCR(46). The observed downmodulation of the endogenousabTCR upon introducing a tumor-specific immune receptorpaves the way towards the broader applicability of our strategy:the combination of siRNA (22), ZFNs (20), or TALENS (23) toknockdown endogenous TCRs with the introduction of otherinnate receptors followed by the depletion with anti-abTCR

beads. This would yield cellular end products with pure popu-lations of engineered cells that do not express their endogenousreceptor and significantly increase the efficacy and safety bothin an autologous and allogeneic scenario. However, our datastress also that a complete long-term interference with endog-enous abTCR chains is most likely not necessary for safety andefficacy. Residual expression of endogenous TCR chains mightbe even needed for an optimal long-term homeostatic prolif-eration (47).

Surprisingly, we also observed a relatively low allo-reactivityin nonengineered bystander cells, questioning whether ourread-out systems have been sensitive enough. We excluded apreferential transduction of na€�ve T cells and subsequent down-regulation of endogenous abTCRs in na€�ve T cells, which havebeen reported as more potent GVHDmediators than memory Tcells (48, 49). However, our observation is also in line with therecent clinical observation that the transfer of nonselected CAR-engineered donor T cells with intact endogenous abTCR chainsin human did not associate with substantial GVHD (50) andmight reflect a rather reduced abTCR expression in such exten-sively cultured and rapidly expanded cells prior infusion. Inline with this observation and in contrast to reports by others(11, 20), we did not observe substantial GVHD in differenthumanized mouse models after adoptive transfer of engineeredimmune cells despite the fact that some models have employedsimilar mouse strains (refs. 11 and 20; T. Straetemans and J.Kuball; unpublished data).

All together, we provide a novel, feasible, and cost-effectivestrategy that is ready to be translated into a GMP-grade pro-cedure to generate untouched engineered immune cells bytaking advantage of an individualized expression system incombination with a strong competitor for endogenous abTCRchains and clinical-grade abTCR beads. We also demonstratethat the interference with endogenous abTCR chains does notnecessarily need to be complete in terms of long-term com-petition in order to obtain a safer and more efficient product.We conclude that the developed depletion strategy is thereforeapplicable to nearly any engineered immune product interfer-ing temporarily or permanently with endogenous abTCRchains.

Disclosure of Potential Conflicts of InterestJ. Kuball reports receiving commercial research grants from Miltenyi and

is listed as an inventor on a patent, owned by University Medical CenterUtrecht, on the use of antibodies for enrichment of engineered T cells withexogenous immune receptors and antibodies for use in depletion of engi-neered T cells. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: T. Straetemans, C. Gr€under, J. KuballDevelopment of methodology: T. Straetemans, C. Gr€under, I. Slaper-Corten-bach, J. KuballAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Straetemans, S. Heijhuurs, S. Hol, J. KuballAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Straetemans, C. Gr€under, S. Heijhuurs, S. Hol,J. KuballWriting, review, and/or revision of the manuscript: T. Straetemans,C. Gr€under, I. Slaper-Cortenbach, H. B€onig, Z. Sebestyen, J. KuballAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Hol, H. B€onigStudy supervision: T. Straetemans, J. Kuball


Straetemans et al.




AcknowledgmentsThe authors thank Kirsten Scholten for assistance in preparation of

experiments.

Grant SupportFunding for this study was provided by ZonMW 43400003 and VIDI-

ZonMW 917.11.337, KWF UU 2013-6426 and UU 2014-6790, and Vrienden

van het UMCU to J. Kuball. Research support was provided by MiltenyiBiotec.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 6, 2014; revised April 12, 2015; accepted May 4, 2015;published OnlineFirst May 19, 2015.

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Straetemans et al.




2015;21:3957-3968. Published OnlineFirst May 19, 2015.Clin Cancer Res Trudy Straetemans, Cordula Gründer, Sabine Heijhuurs, et al. CancerUntouched GMP-Ready Purified Engineered Immune Cells to Treat

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untouched gmp-ready puriﬁed engineered immune cells to ...cancer therapy: preclinical untouched...

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