Bioengineering T cells to target carbohydrate to treat ... · Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection Pappanaicken R. Kumaresana,1, Pallavi

Bioengineering T cells to target carbohydrate to treatopportunistic fungal infectionPappanaicken R. Kumaresana,1, Pallavi R. Manuria,1, Nathaniel D. Albertb, Sourindra Maitia, Harjeet Singha, Tiejuan Mia,Jason Roszika, Brian Rabinovicha, Simon Olivaresa, Janani Krishnamurthya, Ling Zhanga, Amer M. Najjarc,M. Helen Hulsa, Dean A. Leea,d, Richard E. Champline, Dimitrios P. Kontoyiannisb, and Laurence J. N. Coopera,d,2

aDivision of Pediatrics, bDepartment of Infectious Diseases, cDivision of Diagnostic Imaging, Department of Cancer Systems Imaging, dThe University of TexasGraduate School of Biomedical Sciences, and eStem Cell Transplantation and Cell Therapy, University of Texas MD Anderson Cancer Center, Houston,TX, 77030

Edited by Philippa Marrack, Howard Hughes Medical Institute, National Jewish Health, Denver, CO, and approved June 10, 2014 (received for review July8, 2013)

Clinical-grade T cells are genetically modified ex vivo to expresschimeric antigen receptors (CARs) to redirect their specificity totarget tumor-associated antigens in vivo. We now have developedthis molecular strategy to render cytotoxic T cells specific for fungi.We adapted the pattern-recognition receptor Dectin-1 to activateT cells via chimeric CD28 and CD3-ζ (designated “D-CAR”) uponbinding with carbohydrate in the cell wall of Aspergillus germ-lings. T cells genetically modified with the Sleeping Beauty systemto express D-CAR stably were propagated selectively on artificialactivating and propagating cells using an approach similar to thatapproved by the Food and Drug Administration for manufacturingCD19-specific CAR+ T cells for clinical trials. The D-CAR+ T cellsexhibited specificity for β-glucan which led to damage and inhibi-tion of hyphal growth of Aspergillus in vitro and in vivo. Treat-ment of D-CAR+ T cells with steroids did not compromiseantifungal activity significantly. These data support the targetingof carbohydrate antigens by CAR+ T cells and provide a clinicallyappealing strategy to enhance immunity for opportunistic fungalinfections using T-cell gene therapy.

T-cell therapy | β-1,3-glucan | fungus | adoptive immunotherapy

Opportunistic invasive fungal infections (IFI) by Aspergillusspp. cause morbidity and mortality in immunocompromised

patients. Mortality rates associated with invasive Aspergillus (IA)are 22% in patients receiving solid-organ transplants and60–85% in patients receiving hematopoietic stem cell transplants(HSCT) (1, 2). Antifungal agents such as polyenes, triazoles, andechinocandins can be rendered ineffective by suboptimal hostimmunity, emergence of drug-resistant strains, and attendanttoxicities in the recipients (3, 4). Thus, new approaches fortreating IAs are needed. Because the adoptive transfer of ge-netically modified T cells expressing CD19-specific chimericantigen receptors (CARs) has resulted in successful treatment ofpatients with B-cell malignancies (5–9), we sought to determineif a CAR could be developed to redirect T-cell specificity toAspergillus.Among immunocompetent individuals, intact innate immunity

can prevent and eradicate IFI. Endogenous alveolar macro-phages recognize, phagocytize, and kill fungal spores (10), andneutrophils can recognize and eliminate germinating hyphae(11). Immunotherapeutic strategies such as adoptive transfer ofpreselected CD4+ T-cell clones can help control Aspergillus in-fection indirectly by producing interferon-gamma (IFN-γ) (12).An immunotherapeutic strategy deploying cytotoxic T cellsappears to be clinically advantageous, because such cells have anendogenous ability to kill, be propagated to large numbers exvivo, be genetically modified to recognize desired target anti-gens, and contribute to immunologic memory (8).We previously have redirected the specificity of genetically

modified T cells via the stable introduction of a CD19-specificsecond-generation CAR (8, 9). Recognition of CD19 was ach-ieved by a CAR exodomain composed of a CD19-specific single-

chain variable fragment attached to the T-cell surface via amodified IgG4 hinge and fragment-crystallized (Fc) region(13). CAR-dependent and MHC-independent T-cell activationcan be achieved through the CAR endodomain composed ofCD3-ζ and CD28 (14, 15). We modified this prototypical CARdesign to achieve recognition of carbohydrate by accommodatingthe pattern-recognition properties of Dectin-1 (16, 17). Thisdectin is a type II transmembrane protein expressed on macro-phages, neutrophils, and dendritic cells (18) and is specific forβ-glucans, which are glucose polymers consisting of β-1,3-glucanand β-1,6-glucan expressed on the cell wall of fungi (19). BecauseDectin-1 mediates recognition of Aspergillus fumigatus (20), wehypothesized that the extracellular portion of Dectin-1 couldbe adapted as the specificity domain for a CAR (designated“D-CAR”) on T cells to redirect their specificity for this fungus.We report that T cells can be genetically modified by using the

Sleeping Beauty (SB) transposon/transposase system to expressD-CAR stably and can be propagated selectively on artificialactivating and propagating cells (aAPCs). We demonstrate thatthe D-CAR+ T cells (i) bind specifically to laminarin (which isrich in β-1,3-glucan), (ii) exhibit a central memory phenotype,(iii) inhibit growth of Aspergillus hyphae even in the presence of

Significance

Patients with compromised T-cell function are at risk for op-portunistic fungal infections. We have developed a novel ap-proach to restore immunity by using a fungal pattern-recognitionreceptor Dectin-1 to redirect T-cell specificity to carbohydrateantigen in the fungal cell wall. We did so by genetically modifyingT cells using the nonviral Sleeping Beauty gene-transfer systemto enforce expression of a chimeric antigen receptor (CAR) thatrecapitulates the specificity of Dectin-1 (D-CAR). The D-CAR+ Tcells can be electroporated and propagated on artificial activatingand propagating cells in a manner suitable for human application,enabling this immunology to be translated into immunotherapy.This approach has implications for genetically modifying T cells toexpress CARs with specificity for carbohydrate and thus broad-ening their application in the investigational treatment of patho-gens and malignancies.

Author contributions: P.R.K., P.R.M., D.P.K., and L.J.N.C. designed research; P.R.K., P.R.M.,N.D.A., S.M., T.M., J.R., S.O., J.K., and L.Z. performed research; P.R.K. and P.R.M. contrib-uted new reagents/analytic tools; P.R.K., P.R.M., H.S., B.R., J.K., A.M.N., M.H.H., D.A.L., andR.E.C. analyzed data; and P.R.K., P.R.M., D.P.K., and L.J.N.C. wrote the paper.

Conflict of interest statement: L.J.N.C. founded and owns InCellerate, Inc. He has patentswith Sangamo BioSciences with artificial nucleases. He consults with Targazyme, Inc.(formerly American Stem Cells, Inc.), GE Healthcare, Ferring Pharmaceuticals, Inc., andBristol-Myers Squibb. He receives honoraria from Miltenyi Biotec.

This article is a PNAS Direct Submission.1P.R.K. and P.R.M. contributed equally to this work.2To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1312789111/-/DCSupplemental.

10660–10665 | PNAS | July 22, 2014 | vol. 111 | no. 29 www.pnas.org/cgi/doi/10.1073/pnas.1312789111

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dexamethasone, and (iv) target Aspergillus infection in the skinand lung of immunocompromised mice. Thus, using a genetransfer and propagation approach adapted for human applica-tion (21, 22), we have demonstrated that an innate immune re-sponse to fungi can be harnessed by bioengineering cytotoxicT cells. This demonstration provides the foundation for testingwhether D-CAR+ T cells can be infused to improve the survivalof immunocompromised patients who develop opportunistic IAinfection.

ResultsGenerating D-CAR+ Human T Cells. As expected, neither circulatingnor propagated human αβ T cells express Dectin-1 or Dectin-2and thus do not directly recognize Aspergillus using these pattern-recognition receptors. To generate a CAR with the specificity ofDectin-1 capable of activating T cells from peripheral blood, wefused the extracellular domain of human Dectin-1 (23) to ourstandard CAR cassette (15) encoding a modified IgG4 hinge/Fc(13), human CD28 transmembrane and cytoplasmic domains,and the CD3-ζ signaling motif. The resulting D-CAR constructwas subcloned as an SB transposon (Fig. 1A) and electro-trans-ferred with SB11 transposase into peripheral blood-derived hu-man primary T cells (Fig. S1). These data demonstrate that atype II transmembrane receptor can be fashioned into a CAR(containing HA and FLAG epitope tags to validate assembly)and stably expressed on T cells following transposition (Fig.S1B). The propagation of D-CAR+ T cells was not significantly

different from CD19-specific CAR+ T cells cocultured withaAPC (clone #4) (24) that activates/propagates T cells viatransgenic human CD19. The addition of γ-irradiated aAPC(Fig. S2) every 7 d was calculated to support a 6-log numericexpansion (from 106 to 1012) for both D-CAR+ and CD19-spe-cific CAR+ T cells (Fig. 1B). There was no outgrowth of CD3−

cells [e.g., natural killer (NK) cells]. Thirty-five days after elec-troporation, the propagated T cells were stained with mAbspecific for Dectin-1 and analyzed by flow cytometry. Almost all(average 97.6% ± 1.6; n = 3) of the T cells coexpressed CD3 andD-CAR; 90–95% of the propagated D-CAR+ T cells coex-pressed CD8 (Fig. 1C); and, as anticipated, there was no ex-pression of D-CAR on CD19-specific CAR+CD8+ or CAR+

CD4+ T cells. As such, the CD19-specific CAR+ T cells serve asa negative control (hereafter termed “control T cells”). Thesedata demonstrate that D-CAR+ T cells can be expanded nu-merically on aAPCs using an approach similar to that used forthe manufacture of clinical-grade CD19-specific T cells in ourongoing clinical trials.

Genetic Signature and Immunophenotype of D-CAR+ T Cells.We usednCounter analysis of direct digital readouts to quantify mRNAabundance (25), coding for IFN-γ (a Th1 proinflammatorymarker), granzymes A/B (markers of cytotoxicity), DNAX-acti-vating protein (DAP-10) for PI 3-kinase signaling, NK-cell–activating receptor (NKG2D) for costimulation, and zeta-chain–associated protein kinase 70 (ZAP70) and lymphocyte-specificprotein tyrosine kinase (Lck) for T-cell activation. Comparedwith unmodified T cells in peripheral blood, the D-CAR+ T cellsexhibited an increase in levels of IFN-γ (eightfold), granzyme A(94-fold), granzyme B (12-fold), DAP10 (threefold), NKG2D(threefold), Lck (threefold), and ZAP-70 (twofold) (Fig. 1D). Inaddition, there was a 15-fold decrease in killer cell lectin-likereceptor subfamily G, member 1 (KLRG1), which is consistentwith the preservation of T cells that avoid replication senescence(26, 27), and a six- to sevenfold decrease in the nuclear receptorretinoic acid receptor–related orphan receptor α (RORα), whichcontributes to the development of CD4+ Th17 cells (28). Thetherapeutic activity of CD19-specific CAR+ T cells in vivo hasbeen shown to correlate positively with T-cell persistence and isassociated with a central memory (TCM) phenotype (29). Gatingon the CD3+ D-CAR+ subset showed that 96% of the cellscoexpress CD45RO, as is consistent with an outgrowth of mem-ory T cells. Further analysis of the CD45RO+ T cells revealedthat 84% exhibited a TCM immunophenotype on the basis ofcoexpression of CD28, CD62L, and CCR7 (Fig. 1E). These datasuggest that D-CAR+ T cells numerically expanded on aAPCsmay sustain persistence after adoptive transfer with the potentialto provide long-term protection from IA. Moreover, using ourdirect T-cell receptor (TCR) expression analysis (DTEA)method, we found no significant difference in the abundance anddiversity of TCR Vα and Vβ family members expressed by un-modified and genetically modified D-CAR+ T cells (Fig. S3).These data indicate that D-CAR+ T cells emerge from andmaintain a polyclonal population.

Redirected Specificity by D-CAR+ T Cells. Dectin-1 binds to theglucose polymer laminarin that is similar to the sugar moietyfound in fungal walls but does not bind to mannan, the mannosepolymer derived from Saccharomyces cerevisiae (30). D-CAR+ Tcells exhibited the same binding preference, because laminarin,but not mannan, abrogated binding of mAb specific for Dectin-1to D-CAR on genetically modified T cells in a dose-dependentmanner (Fig. 2A). The specificity was evaluated further bycoculturing genetically modified T cells with germinatingAspergillus spores using three in vitro assays. We used microscopyto demonstrate that fungal growth was inhibited significantly byD-CAR+ T cells as compared with CD19-specific CAR+ controlT cells (Fig. 2B). Preincubation of a rabbit polyclonal antibodyraised against the soluble extract of A. fumigatus did prevent theability of D-CAR+ T cells to damage fungal growth, suggesting

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Fig. 1. Bioengineering of D-CAR+ T cells. (A) Components of D-CAR. Theextracellular (Ec) sugar-binding domain of human Dectin-1 was fused inframe to a modified human IgG4 hinge and Fc region and the trans-membrane (Tm) and cytoplasmic (Cyto) domain of human CD28, and CytoCD3-ζ. HA3 and FLAG3 epitope tags were fused on the C and N termini, re-spectively. (B) Numeric expansion of T cells cocultured with aAPC clone #4preloaded with Dectin-1–specific mAb in the presence of soluble IL-2 andIL-21. Data are plotted as mean ± SD from three different donors. Upwardarrows indicate the addition of γ-irradiated aAPC. (C) Expression of D-CARon CD4+ and CD8+ T cells after electroporation and propagation for 35 d.CD19-specific CAR+ T cells from the same donor were used as a gatingcontrol. (D) Comparative gene-expression profiles of the expanded D-CAR+ Tcells and circulating T cells from healthy donors. Abundance of mRNAtranscripts was measured by digital gene expression using the nCounteranalysis system. (E) D-CAR+ T cells propagated for 35 d were costained withanti-CD3 and anti–Dectin-1 mAbs, in addition to staining with anti-CD45RA,CD45RO, CD28, CD62L, and CCR7, and were analyzed by flow cytometry.D-CAR+CD3+CD45RA−RO+ T cells were gated for expression of CD62L, CD28,and CCR7 to define central memory T cells.

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that the redirected specificity is achieved by a specific interactionwith the fungal cell wall. Next, we investigated the capacity ofgenetically modified T cells to kill germinating conidia usinga colorimetric assay to reveal the viability of Aspergillus based onthe ability to reduce a tetrazolium dye (31). Coculture ofD-CAR+ T cells led to an ∼75–80% loss of viability in germi-nating spores compared with a 15–30% loss of viability whencocultured with CD19-specific CAR+ T cells (P < 0.01; Fig. 2C).In the third in vitro assay, we undertook video time-lapsed micros-copy (VTLM) to visualize serially the targeting of germinatingAspergillus by D-CAR+ T cells (Movie S1) and CD19-specificCAR+ T cells (Movie S2) compared with untreated controls(Movie S3). D-CAR+ T cells and background low levels ofCD19-specific T cells bound to germlings (Fig. 2 D, c and g) by3 h. However, by 7 h there was a rapid influx of D-CAR+ T cells(Fig. 2 D, d) but not of control T cells (Fig. 2 D, h). The con-sequence of this binding of D-CAR+ T cells was destruction ofhyphal growth (Fig. 2 D, d) compared with the control T cells(Fig. 2 D, h) and in the absence of T cells (Fig. 2 D, k and l). Themean hyphal length at 24 h was calculated from VTLM toquantify the ability of T cells to destroy hyphae. Compared withcontrol T cells, D-CAR+ T cells reduced hyphal length from 180μm to 70 μm (62% inhibition). In the absence of T cells, hyphaegrew on average to 200 μm over same time period (Fig. 2E).These imaging data support the ability of D-CAR+ T cells totarget the germinating hyphae specifically and as desired, andnot the dormant Aspergillus conidia.

Effect of Dexamethasone on D-CAR+ T Cells. A likely translationalapplication for infusing D-CAR+ T cells will be in recipients

of solid-organ transplants or allogeneic HSCT. Dexamethasone(DEX), a corticosteroid systemically administered for immunosup-pression, has been demonstrated to down-regulate endogenousDectin-1 expression (32) and predisposes patients to fungalinfections (33). We evaluated the effect of DEX on the expres-sion of D-CAR on T cells and found that levels of the introducedimmunoreceptor diminished partially in a dose-dependent man-ner, so that 64% (n = 2) of genetically modified T cells continuedto express D-CAR in 1 μM of DEX. Despite this down-regulationof the introduced immunoreceptor, these T cells retained theirability to damage hyphae (Fig. 3A).

Activation of D-CAR+ T Cells by Aspergillus Germlings. To corrobo-rate the hyphal killing, we evaluated up-regulation of CD107a(LAMP1) that is expressed on the T-cell surface following cy-totoxic degranulation (34). We observed a threefold up-reg-ulation of CD107a on D-CAR+ T cells after coculture withAspergillus germlings (Fig. 3B). Damage to hyphae likely wasmediated by perforin and granzymes, because D-CAR+ T cellsexpressed perforin (92%) and granzyme B (100%) (Fig. 3C).Furthermore, perforin secretion was increased (P < 0.05) inconditioned supernatant after stimulation with aAPCs (clone#4) preloaded with Dectin-1–specific mAb (Fig. 3D). Resultsobtained from our customized bar-coded probe set used tomeasure abundance of mRNA levels suggested that D-CAR+ Tcells produced higher IFN-γ levels than T cells from peripheralblood (Fig. 1D). Therefore, IFN-γ expression was measured inD-CAR+ T cells exposed to germinating Aspergillus, and we alsoobserved a six- to sevenfold increase in IFN-γ protein levels

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Fig. 2. Specificity and damage to Aspergillus hyphae by D-CAR+ T cells. (A)Binding of D-CAR+ T cells by Dectin-1–specific mAb in the absence or pres-ence (200 μg/mL, solid line; 400 μg/mL, dashed line; and 600 μg/mL, dottedline) laminarin or mannan, as evaluated by flow cytometry. Shaded andopen regions in the histograms represent mAb binding in the absence orpresence of sugar, respectively. (B) Preincubation with Aspergillus-specificpolyclonal antibody blocked binding of D-CAR+ T cells to hyphae. Fungalgrowth was inhibited more by D-CAR+ T cells than by CD19-specific CAR+ Tcells. (C) Comparison by XTT assay of the ability of D-CAR+ and CD19-specificCAR+ T cells to damage germinating A. fumigatus hyphae specifically; **P <0.01. (D) D-CAR+ effector (E) T cells were coincubated with targets (T) GFP+

AF293 swollen conidia (E:T ratio of 2:1) and imaged continuously by VTLM.Conidia germlings cocultured with CD19-specific CAR+ T cells and without Tcells served as negative control. (See Movies S1–S3.) Pictures of conidia takenat various time points under fluorescent light (a–l) and with white light (a1–l1) show inhibition of growth by germinating conidia (green fluorescence) byD-CAR+ T cells (a–d), CD19-specific CAR+ T cells (e–h), and in the absence of Tcells (i–l). (Scale bars: 10 μm.) (E) At 24 h, the ability of T cells to inhibit As-pergillus germination as measured by hyphae length obtained from VTLM.Data are shown as mean ± SD. **P < 0.01; n = 3.

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T cells cultured for 1 wk on aAPC preloaded with Dectin-1–specific mAb inthe presence of DEX and soluble IL-2 and IL-21 were analyzed for expressionof D-CAR by flow cytometry (open bars; D-CAR+ T cells cultured in the ab-sence of DEX served as a control) and for their ability to kill A. fumigatushyphae by XTT assay (gray bars; A. fumigatus cultured without T cells servedas control). Data are shown as mean ± SD; n = 2. (B) CD107a expression onD-CAR+ T cells in the absence or presence of A. fumigatus hyphae, analyzedby flow cytometry. Data are shown as mean ± SD; n = 3. (C) Expression ofperforin and granzyme B analyzed by flow cytometry; shaded regions areisotype controls. (D) Perforin secretion in conditioned T-cell culture super-natants after stimulation by aAPCs loaded with Dectin-1–specific mAb. Foldchanges in perforin level were calculated by normalizing against unstimu-lated T cells. (E) D-CAR+ T cells and CD19-specific CAR+ T cells (expressingCD19RCD28) were incubated with germinating A. fumigatus hyphae for 4–6 h.The percentages of D-CAR+IFN-γ+ T cells and CD19-specific CAR+IFN-γ+ T cellswere measured by flow cytometry. T cells stimulated with phorbol12-myr-istate13-acetate/ionomycin served as positive controls. Shown are repre-sentative dot plots from two independent experiments. (F) IFN-γ cytokinelevels were measured in T-cell culture conditioned supernatants after stim-ulation with 50 μg/mL Aspergillus cell lysate.*P < 0.05; **P < 0.01.

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(Fig. 3E). In contrast, no change in IFN-γ levels was observedin control T cells cocultured with germinating Aspergillus. Wealso validated the presence of soluble IFN-γ in conditionedsupernatants of D-CAR+ T cells (P < 0.01) cultured with fungalhyphae extract (Fig. 3F). We detected no secreted IL-17, as isconsistent with decreased levels of mRNA coding for RORα(28). In aggregate, these data demonstrate that electroporatedand propagated D-CAR+ T cells are cytotoxic in response to hy-phae from Aspergillus and can secrete proinflammatory cytokines.

Targeting Aspergillus Infection in Mice by D-CAR+ T Cells. To assessthe in vivo therapeutic efficacy of the D-CAR+ T cells, wetreated immunosuppressed NOD SCID-γ (NSG) mice bearingIA with D-CAR+ T cells. Mice that received no T cells (PBScontrol) or CD19-specific T cells demonstrated Aspergillus in-fection as shown by staining of lung tissue sections with FITC-conjugated Aspergillus-specific antibody. In contrast, treatmentwith D-CAR+ T cells resulted in diminished fungal infection andretention of D-CAR+ T cells (Fig. 4A). Indeed, infused T cellsidentified by phycoerythrin (PE)-conjugated CD3-specific mAbrevealed that fungal hyphae were preferentially covered withD-CAR+ T cells rather than CD19-specific CAR+ T cells. An an-tifungal effect mediated by D-CAR+ T cells was supported by theobservation that the number of visualized genetically modified Tcells, as assessed by red fluorescence, was greater in the mice thatreceived D-CAR+ T cells than in the two controls (P < 0.001;Fig. 4B). In aggregate, quantification of fluorescence showedthat mice receiving D-CAR+ T cells had a significantly lower

pulmonary Aspergillus load than mice treated with control CAR+

T cells or no T cells (P < 0.001; Fig. 4B). This result was sup-ported by measurements of fungal burden in lungs by quantita-tive PCR (qPCR), which revealed that the introduced germlingsunderwent greater damage in the mice receiving D-CAR+ T cellsthan in controls (P < 0.001; Fig. 4C). We extended our obser-vation to another clinically relevant model of IA. Previously, ithas been shown that skin lesions and fungal burden are directlyproportional, and thus the infected surface area is an objectivemeasure of the degree of fungal infection (35). We observed thatsurface area of cutaneous fungal lesions (Fig. 4D) was smaller inmice that received D-CAR+ T cells than in control mice thatreceived CD19-specific CAR+ T cells or no T cells (P < 0.01; Fig.4E). These in vivo data support our in vitro observations thatD-CAR+ T cells can target IA.

Bispecific T Cells Coexpressing D-CAR and CD19-Specific CAR. Patientsat risk for progression of B-cell malignancies, such as patientswith advanced disease undergoing HSCT, are also vulnerable forIA. Therefore, we investigated if the SB system could be adoptedto coexpress CD19-specific CAR and D-CAR in T cells. T cellsthat underwent double transposition were propagated on aAPCclone #4 (36) and were demonstrated by flow cytometry tocoexpress the two CARs (Fig. S4A). To establish the ability ofD-CAR to activate T cells expressing two CAR species, we againdemonstrated that hyphae growth could be targeted on Aspergillusgermlings (Fig. S4B) with damage proportional to the ratio of Tcells to fungal burden (Fig. S4C). Specificity for CD19 was vali-dated by chromium release assay for CD19+ vs. CD19− tumor cells(Fig. S4D). These data demonstrate that T cells can be engineeredto have specificity for both carbohydrate and protein antigens.

DiscussionWe demonstrate a previously unidentified approach for immu-notherapy of Aspergillus based on redirecting T-cell specificitythrough a CAR that recognizes carbohydrate antigen: D-CARwith specificity of Dectin-1 fused to CD28 and CD3-ζ cyto-plasmic signaling domain that delivers a fully competent T-cell–activation signal as defined by killing, cytokine production, andproliferation. We genetically modified primary circulating Tcells using our SB transposon/transposase system that weadapted for human gene transfer. Indeed, we have achievedinstitutional and federal regulatory approvals for four clinical trials(NCT00968760, NCT01497184, NCT01362452, NCT01653717)infusing autologous and allogeneic T cells genetically modifiedwith an SB encoding a CD19-specific CAR and propagationon aAPC (clone #4) (21, 22, 37). To generate clinically relevantnumbers of D-CAR+ T cells for adoptive transfer, T cells wereexpanded numerically on “designer” aAPC. These geneticallymodified T cells coexpress perforin and granzyme and exhibit anability to recognize and lyse Aspergillus germlings. The thera-peutic potential of these T cells is highlighted further by themajority of D-CAR+ T cells (84%) exhibiting a TCM phenotypethat likely can self-renew as well as differentiate into effector Tcells in vivo (38). nCounter Digital profiling was used to ex-amine the abundance of mRNA species; both the transcriptionfactors Eomesodermin and KLRG1 were down-regulated inpropagated D-CAR+ T cells, supporting the observation thatinfused D-CAR+ T cells can sustain proliferation without ter-minal differentiation and avoid replicative senescence (39, 40).Because this is the first time, to our knowledge, that a pattern-

recognition receptor has been adapted to redirect T-cell speci-ficity, we used multiple experiments (fungal cell-killing assay,cytokine production, up-regulation of CD107a, VTLM, and twomouse models) to assess the ability of D-CAR+ T cells to targetgerminating Aspergillus. Based on the results obtained from invitro and in vivo studies, D-CAR+ T cells can directly target andcontrol Aspergillus infection. These assays also demonstrated thatD-CAR is able to activate proinflammatory and cytolytic ma-chinery such as the perforin/granzyme pathway of geneticallymodified T cells. The exocytosis of cytolytic granules apparently

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Fig. 4. Targeting Aspergillus infection in mice by D-CAR+ T cells: (A) Pul-monary Aspergillus infection in NSG mice. Fluorescent microscopic pictures oflung sections of Aspergillus (AF293) in mice that received PBS, CD19-specificCAR+ T cells, and D-CAR+ T cells. (B) Mean fluorescence intensity for stainingwith anti-human CD3-PE and anti-Aspergillus-FITC was calculated using In-form imaging software. Values are shown as mean fluorescent intensity(counts per image objects) from three lung slides per group. ***P < 0.001. (C)Quantification of A. fumigatus burden in lungs of mice 4 d after infection, asdetermined by qPCR. Data are expressed as the number of conidial equivalent(CE) copies of DNA in aliquots of lung homogenates. Data are shown as mean± SD; ***P < 0.001 compared with control mice receiving PBS (labeled “As-pergillus”) or CD19-specific CAR+ T cells; n = 5 mice. (D) Cutaneous Aspergillusinfection in NSG mice. Mice received no therapy, CD19-specific (CD19RCD28)CAR+ T cells, D-CAR+ T cells, and T cells expressing no CAR (No DNA). Site offungal lesions are indicated by arrows. After 96 h, mice were anesthetized,and skin lesions were measured. (E) Cutaneous fungal burden of mice treatedwith D-CAR+ T cells compared with Aspergillus without treatment, CD19-specific CAR+ T-cells, or (c) no DNA electroporated T-cells. *P < 0.05, treat-ment with D-CAR+ cells vs. other three conditions; n = 5.

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can disrupt fungal germination directly, as evident from thereports that granzyme-deficient mice are susceptible to IFI (41,42). The production of IFN-γ from the D-CAR+ T cells mayfurther augment immunity to IFIs, because pharmacological dos-ing of recombinant IFN-γ (43) or of IFN-γ derived from CD4+

helper T cells or NK cells has been shown to augment anti-As-pergillus activity (44, 45). However, it remains to be determinedwhether the D-CAR–dependent production of IFN-γ can con-tribute directly to the clearance of fungal infections or indirectlythrough the activation of granulocytes. Other cytokines, such asIL-17, produced by subsets of T cells also may participate in an-tifungal immunity by activating neutrophils (46). However, we didnot observe IL-17 secretion by D-CAR+ T cells, as is is consistentwith decreased expression of RORα, which plays a critical role inaugmenting IL-17 production from T cells (28, 47).Targeting by D-CAR+ T cells may be augmented by combina-

tion therapies. For example, Aspergillus preexposed to caspofungincan unmask β-glucan residues in the cell wall of fungi and enhanceantifungal activity mediated by neutrophils (31). Engineering aT-cell response to Aspergillus hyphae also may overcome con-genital impairment of the innate immune system, as with theDectin-1 Y238X polymorphism (48). This mutation is associatedwith diminished Dectin-1 receptor activity, increased suscepti-bility to IFI among recipients of HSCT (48), and loss of TLR4-mediated signals during hyphal germination, thereby contribut-ing to the evasion of immune recognition by Aspergillus (49).To help translate our findings to clinical practice, D-CAR+ T

cells must maintain effector function in the presence of system-ically administered immunosuppressive medications. The corti-costeroid DEX can down-regulate expression of Dectin-1 ongranulocytes and thereby increase recipients’ susceptibility toIFI. Although we did observe a decrease in D-CAR expression inelectroporated/propagated T cells exposed to DEX, the level ofD-CAR expression still was sufficient to damage germinatingAspergillus hyphae. This result indicates that adoptive transfer ofD-CAR+ T cells may remain effective in patients at risk for IFIbecause of the systemic administration of DEX. In addition, wegenerated bispecific T cells using the SB system that coexpressCD19-specific CAR (CD19RCD28) and D-CAR. These T cellsexhibited specificity for both CD19 and Aspergillus, raising thepossibility that one population of T cells can be customized totarget relapse and infection, which are two common causes ofmorbidity and mortality after allogeneic HSCT.In summary, we report a bioengineering approach to redirect

the specificity of T cells by the expression of a novel CAR thatincorporates the pattern-recognition ability of Dectin-1 to rec-ognize Aspergillus germlings. The D-CAR design enabled us toadapt an immunoreceptor that operates within the landscape ofinnate immunity for expression in T cells to co-opt their en-dogenous cytolytic machinery to target Aspergillus hyphae. Thisapproach is clinically appealing, because long-lived T cells can begenetically modified (e.g., using the SB system) and propagatedto large numbers (e.g., using γ-irradiated aAPC) ex vivo incompliance with cGMP for Phase I/II trials. One proposedclinical application for the administration of donor-derivedD-CAR+ T cells is administration following allogeneic HSCT,because the introduced D-CAR is capable of recognizing β-glucanmoieties present on opportunistic fungal infections. Furthermore,the recognition of carbohydrate antigens by CAR broadens thetranslational appeal of genetically modified T cells to targetpathogens as well as tumor-associated carbohydrate antigens.

Materials and MethodsPlasmids. D-CAR design was based on our second-generation CD19-specificCAR, CD19RCD28 (15). In brief, the sequence encoding the extracellular (Ec)sugar-binding domain of human Dectin-1 (GenBank accession no. AY026769)was fused to the modified human IgG4 hinge and Fc regions (13), which inturn were combined with the transmembrane and cytoplasmic residues ofhuman CD28 molecule and then the human cytoplasmic CD3-ζ chain. Thehuman codon optimized (CoOp) D-CAR (CoOp D-CAR), synthesized by GeneArt,was fused at the (Ec) C terminus to a triplicate HA epitope (amino acid

sequence YPYDVPDYA) and to the FLAG3 epitope (amino acid sequenceDYKDDDDC) on the N terminus by PCR to obtain FLAG(CoOp)–D-CAR–HA3 (Fig.1A) with flanking 5′ SpeI and 3′ NotI restriction enzyme sites. After sequencevalidation, the FLAG3(CoOp)–D-CAR–HA3 fragment was subcloned into the SBtransposon DNA plasmid CoOpCD19RCD28/pSBSO (used to express CD19RCD28for human application), by restriction digestion at SpeI-NruI and NheI-SmaI sites,thus replacing the CD19RCD28 CAR fragment with the FLAG3(CoOp)–D-CAR–HA3

fragment to create the final plasmid CoOpD D-CAR/pSBSO (designated “pSBD-CAR”). The DNA plasmid pCMV-SB11 expresses the SB11 transposase (21).

Cells. Primary human T cells were isolated by density gradient centrifugationover Ficoll-Paque-Plus (GE Healthcare Bio-Sciences AB) from peripheral bloodobtained from the Gulf Coast Regional Blood Center (Houston, TX) afterinformed consent. All primary cells and cell lines were cultured in RPMImedium 1640 (HyClone Laboratories) supplemented with 2 mM Glutamax-1(catalog no. 35050-061; Life Technologies) and 10% heat-inactivated FBS(HyClone). D-CAR+ T cells were generated as previously described for CD19-specific CAR+ T cells (37). In brief, T cells were electroporated with DNAplasmids from the SB system coding for D-CAR and SB11 and were propa-gated on aAPCs (clone #4) preloaded with Dectin-1–specific mAb in thepresence of IL-2 and IL-21. Bispecific CD19-specific CAR+ T cells coexpressingD-CAR were generated using double transposition whereby the SB systemwas used to coexpress two CARs. Mock-electroporated (“no DNA”) CAR−

T cells were propagated on aAPCs (clone #4) preloaded with CD3-specificmAb OKT3 (catalog no. 16-0037-85; eBioscience) in the presence of IL-2 andIL-21 (50). All cell lines used in this study were authenticated by fingerprinting at the MD Anderson Cancer Center sequencing core facility. Addi-tional details are provided in SI Materials and Methods.

Fungal Isolates, Growth, and Germination. Two A. fumigatus isolates wereused: a reference wild-type strain, A. fumigatus 293 (AF293), and a strain of A.fumigatus expressing GFP (GFP-AF293, GFP+AF), a kind gift from Kieren Marr(The Johns Hopkins University School of Medicine, Baltimore, MD) (51). Bothisolates were grown on potato dextrose agar for 7 d at 37 °C before conidiawere harvested by flooding the plates with PBS containing 0.1% Tween 20(35). For killing assays, conidia were counted using a hemocytometer andresuspended at a concentration of 105 spores/mL in RPMI containing 10% FBS.One milliliter containing 105 spores in Eppendorf tubes was shaken (220 rpm,digital incubator shaker, Innova 4300, New Brunswick Scientific, Inc.) at 37 °Cfor 12–16 h for germination. Similarly, for VTLM, harvested GFP+AF conidiawere incubated and shaken for 4–5 h before analysis.

XTT Assay. The targeting of Aspergillus was assessed using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-sH-tetrazoliumhydroxide (XTT)-based colorimetric assay (31). Germinating conidia wereincubated for 2–3 h with 106 T cells (effector:target ratio, 10:1) in RPMI 1640with 10% FBS. CD19-specific CAR+ T cells were used as a negative control.After incubation, T cells were lysed with cold, sterile water (hypotonic lysis).Damage to germlings was calculated following the manufacturer’s protocol(XTT assay, catalog no. X4251; Sigma).

Animal Studies. All animal experiments were approved by the University ofTexas MD Anderson Cancer Center Institutional Animal Care and Use Com-mittee. Eight-week-old female NSG mice (Jackson Laboratory) weighing 18–20g were used and housed in a sterile facility. Cyclophosphamide (100 or 150mg/kg of body weight) was injected i.p. to achieve immunosuppression 4 dand 1 d before inoculation with Aspergillus (AF293) spores (defined as day 0).Pulmonary fungal infection was induced by infecting with AF293 conidia (1.5 ×106 per mouse) via the sino-pulmonary route (52). The mice were grouped (n =5) into four groups. The first group received no T cells; the second group re-ceived CD19-specific CAR+ T cells; the third group received D-CAR+ T cells; andthe fourth group received T cells that did not express CAR (“no DNA”). T cellswere dosed at 2 × 107 by i.v. injection every day for 3 days beginning on day 0.All surviving mice were killed on day 4 to determine fungal burden. The sameexperimental design was used to establish a cutaneous fungal infection, ex-cept that T cells were administered s.c. into the same site at a 1:10 ratio ofAF293 conidia (1.5 × 106) per mouse) to T cells (15 × 106 per mouse). Skinlesions were measured daily using digital calipers (Fisher Scientific), and thelongest and shortest diameters were recorded (35).

Statistics. Graphs were plotted using Prism v. 4.0 software (Graph PadSoftware). Statistical analysis was carried out using SAS 9.3 (SAS Inc.). A two-tailed P value of <0.05 was considered statistically significant. Skin lesionareas and fungal burden were compared among groups of mice using anunpaired Student t test.

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ACKNOWLEDGMENTS. We thank the flow cytometry and cellular imaging corefacility at the MD Anderson Cancer Center (MDACC) [supported by MDACCCancer Center Support Grant (CCSG) CA016672]; Dr. Perry Hackett (Universityof Minnesota) for assistance with the SB system; Dr. Kieren Marr (The JohnHopkins University) for helping develop the concept; Dr. Judy Moyes (MDACC)for editing; Dr. GeorgeMcNamara (MDACC) for help with image processing; andDr. Jianlang Dai (MDACC) for statistical analysis. This work was supported byCancer Center Core Grant CA16672; National Institutes of Health (NIH) GrantsR01 CA124782, CA120956, CA141303, and CA141303; NIH Grant R33 CA116127;NIH Grant P01 CA148600; the Burroughs Wellcome Fund; the Cancer Prevention

and Research Institute of Texas; the Chronic Lymphocytic Leukemia Global Re-search Foundation; the Defense Advanced Research Projects Agency (DefenseSciences Office); the Department of Defense; the estate of Noelan L. Bibler; theGillson Longenbaugh Foundation; the Harry T. Mangurian, Jr. Fund for LeukemiaImmunotherapy; the Institute of Personalized Cancer Therapy; the Leukemia andLymphoma Society; the Lymphoma Research Foundation; MDACC’s Sister Insti-tution Network Fund; the Miller Foundation; Mr. Herb Simons; Mr. and Mrs. JoeH. Scales; Mr. Thomas Scott; the National Foundation for Cancer Research; thePediatric Cancer Research Foundation; and the William Lawrence and BlancheHughes Children’s Foundation.

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