Augmentation of CAR T-cell Trafficking andAntitumor Efficacy by Blocking Protein Kinase ALocalizationKheng Newick1, Shaun O'Brien1, Jing Sun1, Veena Kapoor1, Steven Maceyko1,Albert Lo2, Ellen Pur�e2, Edmund Moon1, and Steven M. Albelda1
Abstract
Antitumor treatments based on the infusion of T cells expres-sing chimeric antigen receptors (CAR T cells) are still relativelyineffective for solid tumors, due to the presence of immunosup-pressive mediators [such as prostaglandin E2 (PGE2) andadenosine] and poor T-cell trafficking. PGE2 and adenosineactivate protein kinase A (PKA), which then inhibits T-cell recep-tor (TCR) activation. This inhibition process requires PKA tolocalize to the immune synapse via binding to the membraneprotein ezrin. We generated CAR T cells that expressed a smallpeptide called the "regulatory subunit I anchoring disruptor"(RIAD) that inhibits the association of PKA with ezrin, thusblunting the negative effects of PKA on TCR activation. Afterexposure to PGE2 or adenosine in vitro, CAR-RIAD T cells showedincreased TCR signaling, released more cytokines, and showed
enhanced killing of tumor cells compared with CAR T cells. Wheninjected into tumor-bearing mice, the antitumor efficacy ofmurine and human CAR-RIAD T cells was enhanced comparedwith that of CAR T cells, due to resistance to tumor-inducedhypofunction and increased T-cell infiltration of establishedtumors. Subsequent in vitro assays showed that both mouse andhuman CAR-RIAD cells migrated more efficiently than CAR cellsdid in response to the chemokine CXCL10 and also had betteradhesion to various matrices. Thus, the intracellular addition ofthe RIAD peptide to adoptively transferred CAR T cells augmentstheir efficacy by increasing their effector function and by improv-ing trafficking into tumor sites. This treatment strategy, therefore,shows potential clinical application for treating solid tumors.Cancer Immunol Res; 4(6); 541–51. �2016 AACR.
IntroductionThe adoptive transfer of T cells transfected with chimeric
antigen receptor (CAR) genes is showing great promise in treatingbloodborne tumors (1–3). However, in the case of solid tumors,many immunosuppressive factors exist within the tumor micro-environment (TME) that appear to render these CAR T cellsineffective (4–10). Adenosine, a purine nucleoside present athigh concentrations during hypoxia (11), and prostaglandin E2(PGE2), a smallmolecule derivative of arachidonic acid producedby the inducible cyclooxygenase 2 enzyme (COX2), are potentinhibitors of T-cell proliferation and activity via signaling throughtheir own G-coupled receptors to activate protein kinase A (PKA)in a cyclic AMP (cAMP)–dependent manner (12–17). PKA loca-lizes to the immune synapse and then affects multiple proteins inthe T-cell signaling cascade (18–20). One of the most importantand proximal effects is the phosphorylation of serine-364 on the
kinase Csk, resulting in activation. Activated Csk then phosphor-ylates the key signaling molecule Lck on tyrosine-505, whichinhibits its activity. This leads to the subsequent inhibition ofT-cell signaling and impaired T-cell receptor (TCR)–induced T-cellproliferation and cytotoxic ability. The activation state of Cskappears to be important in setting the TCR signaling thresholdand affinity recognition (21).
The PKA holoenzyme is a heterotetramer consisting of tworegulatory (R) subunits that can further be categorized into type IPKA (consisting of RI subunits) and type II PKA (consisting of RIIsubunits), and two catalytic (C) subunits (19). After binding tocAMP, the R subunits dissociate, and the C subunits proceed tophosphorylate a myriad of target substrates. Given the negativeeffects of PKA activation described above, much effort has beeninvested in manipulating the PKA pathway in T cells to improvetumor killing, including the generation of dominant-negativePKA constructs (22–24), the use of receptor agonists/antagonists(25–27), andPKA inhibitors (28), aswell as geneticmanipulationof the PKA system in various mouse models (12).
An alternative inhibitory approach takes advantage of the factthat in order for PKA to elicit its functions, it must be tethered tolipid rafts in close proximity to the cAMP-generating enzymeadenylyl cyclase. This spatial regulation is mediated by so-called"A-kinase anchoring proteins (AKAP)" that serve as a platformwhere cAMP and PKA signaling converge (29–31). Over 50distinct AKAPs have been identified (reviewed in ref. 32). In2007, Ruppelt and colleagues described the requirement of theAKAP ezrin in tethering PKA to the lipid rafts in T cells (30).
Based on this observation, peptides that bind to the RI subunitof PKA with high affinity and disrupt PKA–AKAP associations,
1Department of Medicine, Perelman School of Medicine, University ofPennsylvania, Philadelphia, Pennsylvania. 2Department of BiomedicalSciences, School of Veterinary Medicine at the University of Pennsyl-vania, Philadelphia, Pennsylvania.
Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).
Corresponding Author: Steven M. Albelda, Perelman School of Medicine at theUniversity of Pennsylvania, Abramson Research Center, 3615 Civic CenterBoulevard, Philadelphia, PA 19104. Phone: 215-573-9933; Fax: 215-573-4469;E-mail [email protected]
including the ezrin–PKA association, were designed (29, 31).One peptide, called "regulatory subunit I anchoring disruptor"(RIAD), displaced PKA from lipid rafts and ultimately dimin-ished phosphorylation of Y505 on Lck, leading to upregulatedT-cell signaling (31). Later, an endogenous element upstreamin the traditional A-kinase binding domain of ezrin thatenhanced RIAD binding to PKA called the "RI specifier region,"or RISR was identified (29). A transgenic mouse model in whichRISR–RIAD was expressed in T cells under the control of thedistal lck promoter was generated, and these mice exhibitedheightened TCR signaling and IL2 secretion, and resistance toPGE2 and murine AIDS (33).
Given the key role of PKA signaling in the inhibition of T-cellfunction in tumors and our ability to genetically manipulateT cells for adoptive transfer, we hypothesized that cloning theRISR–RIAD transgene (referred to as RIAD henceforth; ref. 34)into T cells also expressing a CAR would enhance their functionwithin the TME and result in superior tumoricidal ability ascompared with CAR T cells alone.
Materials and MethodsOverall experimental design
The peptide blocking the localization of PKA to the immu-nologic synapse (RISR–RIAD) was cloned into retroviral andlentiviral vectors encoding CARs directed against humanmesothelin (mesoCAR) or murine fibroblast activation protein(FAP). Murine and human T cells expressing human CAR(mmesoCAR and hmesoCAR, respectively) and CAR-RIAD(mmesoCAR-RIAD and hmesoCAR-RIAD, respectively) con-structs were evaluated for their in vitro and in vivo functions.All experiments were performed at least thrice in independentfashions, unless otherwise indicated.
Generation of RIAD-expressing mesoCAR and FAPCAR andT-cell production
The RISR–RIAD construct (32–34), into which Myc andDDK (FLAG) tags were incorporated, was synthesized byIntegrated DNA Technologies in the pIDT.SMART cloningplasmid. The insert was subcloned into CAR constructs inretroviral vectors that were used to transduce mouse T cells(35) and into lentiviral vectors (36) for use in human T cellsas previously described. The structure of these constructs isshown in Supplementary Fig. S1. The isolation, bead activa-tion, transduction and subsequent expansion of primaryhuman or mouse T cells were carried out as previously des-cribed (10, 35).
The transduction efficiency with either mesoCAR or meso-CAR-RIAD cells was always checked after transduction andbefore each experiment. Equal numbers of mesoCAR versusmesoCAR-RIAD cells were always used for each in vitro killing/cytokine experiment (Fig. 1), in vitro migration assays (Fig. 5),and in vivo experiments (Fig. 3).
AnimalsAll animal protocols were approved and carried out in com-
pliance with the Institutional Animal Care and Use Committee(IACUC) at the University of Pennsylvania. Studies using retro-viral MigR1-transduced T cells were carried out in wild-typeC57BL/6 (strain CD45.2) mice obtained from Charles RiverLaboratories. In some studies, murine T cells were prepared from
CD45.1C57BL/6mice. Studies using lentiviral pTRPE-transducedhuman T cells were conducted in NOD/scid/IL2rg�/� (NSG)micebred at the Children's Hospital of Philadelphia. All test animalsused were females at 10 to 12 weeks of age.
Cell linesAll cell lines used were cultured as described (10) and were
routinely examined for Mycoplasma infection. AE17ova murinemesothelioma cells were obtained from the University ofWestern Australia (37), whereas epithelial mesothelioma (EM)human mesothelioma cells were derived from a patient's tumor(called EMparental, or EMP; ref. 10). The SS1 scFv used in themesothelin CAR is specific against human mesothelin, andtherefore human mesothelin was stably transduced into themurine AE17ova (AE17meso) and human EM (EMmeso)mesothelioma cancer cell lines (10, 35). The murine 4662pancreatic ductal carcinoma cell line (PDA4662) was derivedfrom an autochthonous pancreatic tumor isolated from a fullybackcrossed C57BL/6 KrasG12D:Trp53R172H:Pdx-1 Cre (KPC)mouse (13). Cell lines were not authenticated.
AntibodiesThe following conjugated antibodies for flow cytometric
analysis of murine cells were purchased from Biolegend: CD8(#100762), CD4 (#100406), IFNg (#505825), IL2 (#503808),and anti-GFP (#338008). For human cells, the following werepurchased from Biolegend: FOXP3 (#320106); BD Biosciences:CD45 (#555483), CD8 (#555367), IL2 (#340448), CD69(#555530), IFNg (#562016), and TNFa (#340511); and R&DBiosystems: human mesothelin (FAB32652P). For the detec-tion of live cells, Aqua Live/Dead (Life Technologies #L34957)was used.
PGE2 ELISATumors and normal lung and liver were dissected from mice,
snap frozen in liquid nitrogen, and homogenized in cold 1XPBS containing a cocktail tablet of protease inhibitors (Roche#04693116001). Homogenates were sonicated and centri-fuged, and supernatants were used for PGE2 ELISA (Abcam#ab133021).
Assessment of T-cell effector functionsFunctional assays performed to characterize persistence and
activity of endogenous and genetically modified T cells are out-lined below.
In vitro cytotoxicity and IFNg ELISA. Triplicate wells of 5,000luciferase-expressing parental and antigen-expressing cellswere cocultured with differing ratios of CAR-expressingT cells to tumor cells as previously described (10). Cytotoxi-city of T cells was evaluated the following day, and culturesupernatants were collected for IFNg ELISA (10, 35, 36). Toevaluate the resistance of RIAD-expressing T cells to immuno-suppression, in vitro cytotoxicity assays were also performedin the presence of PGE2 (Enzo Life Sciences; #BML-PG007)and adenosine (Sigma; #A9251).
In vivo studies. For mesoCAR and FAPCAR studies in wild-typeC57BL/6 mice, 2�106 AE17meso or PDA4662 cells were sub-cutaneously inoculated (35, 36). Similarly, for mesoCAR
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studies in immunodeficient NSG mice, 2�106 EMmeso cellswere subcutaneously inoculated (10). When tumors wereapproximately 200 mm3, mice were given 107 CAR-expressingT cells via intravenous administration (10, 35, 36). Tumorvolume was monitored by caliper measurements twice weekly,and at different time points, tumor-bearing mice were sacrificedfor mechanistic studies using immunoblotting and/or flowcytometry. All experiments had a minimum of 5 mice pergroup and were performed at least three times.
Immunoblotting. The phosphorylation status of various proximalT-cell signaling entities was assessed by immunoblotting (pERK,pAkt, pLck) using CAR T cells and plate-bound CD3/CD28 anti-bodies (35). T cells were also exposed tomesothelin-coated beadsas described (35) and harvested after 20 minutes. Additionally,antibodies for the detection of pLckY505 were purchased fromCell Signaling Technologies (#2751S) and pCskS364 from Abcam(#ab61782).
Ex vivo T-cell analysis. Tumors were harvested from mice, micro-dissected, digested, andused in ex vivo tumor assays asdescribed indetail in previous publications (10, 35, 36).
Fluorescence-activated cell sorting. Single-cell suspensions werestained for surface and intracellular markers using the previouslylisted antibodies based on the manufacturer's recommendations.For intracellular cytokine staining, cells were stimulated for 4 to 6hours at 37�C in the presence of 0.7 mg/mL GolgiStop (BDBiosciences #554724) with plate-bound 1 mg/mL anti-CD3and/or 2 mg/mL anti-CD28 antibodies, and 30 ng/mL PMA and1 mmol/L ionomycin. Acquisition was performed on a CyAn-ADPAnalyzer (Beckman Coulter) or a BD LSRFortessa (BD Bios-ciences). Data were analyzed using FlowJo (TreeStar).
Transwell migration studies. An equal number of transducedmesoCAR-GFP and mesoCAR-RIAD-mCherry cells were placedin 0.5-mm polycarbonate Transwell membranes and allowed to
Figure 1.Primary T cells transduced with mesoCAR-RIAD exhibit superior killing ability and robust IFNg production in vitro and are resistant to adenosine and PGE2suppression. A, human mesoCAR and mesoCAR-RIAD T cells were cocultured at various E:T ratios with parental EM (EMP) or mesothelin-expressing EM (EMmeso)cells (left). Murine mesoCAR and mesoCAR-RIAD T cells were cultured with ova- or mesothelin-expressing AE17 murine mesothelioma cells (right). Thesetumor cells are also stably transduced with luciferase. After overnight incubation, the number of live tumor cells was determined by quantifying luciferase activity.B, cell culture supernatants from the assay described above were analyzed for IFNg production via ELISA. Left, IFNg production by human T cells; right, IFNgproduction by murine T cells. C, mesoCAR and mesoCAR-RIAD human T cells were cocultured with EMmeso cells [Effector:Target (E:T) 10:1] overnight in thepresence of increasing doses of adenosine or PGE2 (left). Similarly, the coculture assay was performed for murine mesoCAR and mesoCAR-RIAD T cells andAE17meso tumor cells at an E:T ratio of 5:1; right). Statistical analyses were performed using one-way ANOVA comparing mesoCAR and mesoCAR RIADcells. � , P � 0.05; �� , P � 0.01; ��� , P � 0.001. At least three independent experimental replicates were performed. Data represent means � SEM; n ¼ 3 replicatesper condition.
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migrate toward PBS, cell culture medium alone, or with differentconcentrations of the chemokine CXCL10 in tissue culture plates.After 4 hours, migrated cells in the bottom wells were collectedand were counted based on GFP and mCherry expression.
Adhesion assays. Nontreated 24-well tissue culture plates werecoated with 5 mg/mL of fibronectin, ICAM1, and VCAM1 inPBS for 2 hours at room temperature. These substrates wereremoved prior to rinsing with PBS. Plates were then blocked withcomplete cell culture medium for 30 minutes at 37�C. An equalnumber of transduced mesoCAR-GFP and mesoCAR-RIAD-mCherry T cells were cultured overnight with these substrates.The next day, cells were harvested and counted based on GFPand mCherry expression.
Statistical analysisAll results were reported as means � SEM. For studies com-
paring two groups, the Student t test was used, while for studiescomparing more than two groups, one- or two-way ANOVAwas used with the appropriate post hoc testing, with �, P � 0.05;��, P � 0.01; ���, P � 0.001; and ����, P � 0.0001.
ResultsRIAD enhances in vitro CAR T-cell function
CAR T cells expressing the scFv from anti-human mesothelin,along with the human CD3z and 4-1BB cytoplasmic domains(mesoCAR), slow mesothelin-expressing tumors in mice (10, 35,36, 38, 39), but are limited by T-cell hypofunction induced by animmunosuppressive solid TME (10). To disarm one of the pos-sible inhibitory elements (the cAMP–PKA pathway), we usedmodified lentiviruses (for human T cells) and retroviruses (formurine T cells) to express mesoCAR, with and without the RIADtransgene (Supplementary Fig. S2). The transduction efficiencywith either mesoCAR or mesoCAR-RIAD was checked after trans-duction and before each experiment. For human T cells, this wasbetween 35% and 60%. For mouse T cells, this was between 60%and 88%.
The baseline characteristics of the human andmousemesoCART cells versus the mesoCAR-RIAD T cells showed no consistentdifferences with regard to the CD4:CD8 ratio, differentiationphenotype, or expression of activation or inhibitory receptors.Details are provided in Supplementary Figs. S3 and S4.
In order to assess the effector functions of these T cells, trans-duced T cells were cocultured overnight with human and murinemesothelioma cells expressingmesothelin. Comparedwith T cellstransduced with mesoCAR alone, both human- and murine-transduced mesoCAR-RIAD T cells generally showed enhancedkilling ability (Fig. 1A) and higher IFNg production in a dose-dependentmanner (Fig. 1B). The constructswere target specific, aslittle to no killing of tumor cells that did not express the targetmesothelin was observed.
To further investigate the functionality and activity of meso-CAR-RIAD T cells at baseline, we stimulated human T cells withhuman mesothelin protein coated on beads or PMA/ionomycin(Supplementary Fig. S5) and performed flow cytometry. Thecytokine release at baseline andafter bead stimulationwas slightlyhigher in mesoCAR-RIAD T cells versus mesoCAR T cells (aftermeso-bead simulation, we saw 8.2% of mesoCAR-RIAD cellsmaking IFNg vs. only 2.4% of mesoCAR T cells). However, insimilar experiments using murine T cells transduced with meso-
CAR vs. mesoCAR-RIAD T cells, we saw no differences in cytokinerelease after mesothelin bead exposure (data not shown).
MesoCAR-RIAD T cells were more resistant to immunosup-pression mediated by adenosine and PGE2. An overnight cocul-ture assay was performed with both human (Fig. 1C, left) andmurine (Fig. 1C, right) T cells in the presence/absence of varyingdoses of adenosine and PGE2. As expected, we observed a dose-dependent inhibition of tumor-cell killing by mesoCAR T cells inthe presence of adenosine or PGE2. However, killing by themesoCAR-RIAD T cells was virtually unaffected by these inhibi-tory molecules [data shown at 5:1 effector-to-target ratio (E:T) forhuman cells, and 10:1 E:T for murine cells].
TCR signaling in mesoCAR-RIAD T cellsTo evaluate signaling, both human and murine CAR T cells
(50% transduced cells) were examined at baseline and after 20minutes of exposure to plate-bound anti-CD3 and anti-CD28antibodies. In human cells (Fig. 2A, left), mesoCAR-RIAD Tcells showed some evidence of basal activation with increasedphosphorylation of LckY394 (an activated form of Lck) and Aktcompared with mesoCAR T cells. After stimulation, in bothtypes of cells, we observed the expected increases in Erk,LckY394, and Akt phosphorylation. In mesoCAR-RIAD T cells,slightly higher levels of phosphorylation of these moleculeswere observed. Densitometry analyses are shown in Supple-mentary Fig. S6.
With human T cells, we were also able to obtain immunoblotsafter stimulating the cells with mesothelin-coated beads to spe-cifically assess CAR-mediated pERK generation (Fig. 2A, right).Again,we sawa small increase inbasal pERK levels (compare lanes1 and 2). Exposure to the beadsmodestly increased pERK levels inmesoCAR T cells (compare lanes 1 and 3). However, higher pERKlevels were seen after bead exposure of mesoCAR-RIAD T cells(compare lanes 2 and 4, and lanes 3 and 4).
In murine T cells (Fig. 2B), there appeared to be a slightupregulation of pAkt at baseline. After CD3/CD28 activation,pERK increased inmesoCAR-RIAD T cells versusmesoCAR T cells,but no increase in pAkt was observed. For unclear reasons, studiesusing mesothelin-coated beads were not successful in murineT cells.
PKA inhibitory signaling attenuated in mesoCAR-RIADT cells
PKA regulates T-cell signaling by phosphorylating the kinaseCsk at S364, which leads to phosphorylation of the key TCRproximal signaling molecule, Lck, at Y505, a change that inhibitsthe activity of Lck. To confirm that this mechanism of RIADinactivation was operative in our cells, we assessed the phosphor-ylation status of CskS364 and LckY505 in both human and mouseCAR T cells (Fig. 2C); both groups consisted of�50% transducedT cells. As predicted, we observed a reduction of phosphorylationat both these residues at baseline in mesoCAR-RIAD T cellscompared with mesoCAR T cells.
Enhanced tumor killing by mesoCAR-RIAD T cells in vivoTo compare the ability of mesoCAR-RIAD T cells versus meso-
CAR T cells to control tumor burden, we used two tumor modelsthat have producemore PGE2 comparedwith normal lung or livertissue (Supplementary Fig. S7A). Human EMmeso cells growingin the flanks of immunodeficient NSGmice were administered in
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a single intravenous dose of 107 mesoCAR- or mesoCAR-RIAD–
expressing primary human T cells when the tumors were �200mm3 in size. Animals were sacrificed 32 days after CAR T-cellinjection to allow analysis of tumor-infiltrating lymphocytes (TIL;see later). Although we saw clear antitumor activity, neither typeof CAR T cell could cure these established tumors (Fig. 3).EMmeso-bearing NSGmice treated withmesoCAR T cells showedsignificantly slower tumor progression by approximately 40%(P � 0.01) compared with untreated tumors; however, injectionof mesoCAR-RIAD T cells significantly enhanced the mesoCARantitumor effect (P� 0.0001), resulting in tumors that were 70%smaller compared with untreated tumors (Fig. 3A). mesoCAR-RIAD T cells significantly reduced the growth of EMmeso tumorscompared with mesoCAR T cells, P � 0.05 (Fig. 3A).
Murine AE17meso cells were injected subcutaneously in theflanksofwild-typeC57BL/6mice, andafter tumorswere established(about 7–10 days after inoculation), a single dose of 107mesoCARor mesoCAR-RIADmurine T cells was administered intravenously.Tumor growth was monitored for the next 10 to 14 days. Again,we observed some tumor slowing by mesoCAR T cells, but meso-CAR-RIAD T cells significantly reduced the growth of AE17mesotumors compared with mesoCAR T cells, P � 0.05 (Fig. 3B).
The use of adoptively transferred RIAD-expressing cells didnot nonspecifically boost endogenous antitumor activity ofmouse T cells, as mesoCAR-RIAD T cells were ineffective incontrolling AE17ova tumor (which do not express the cognateantigen mesothelin) progression in wild-type mice (Supple-mentary Fig. S7B).
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Figure 2.RIAD attenuates PKA signaling and enhances TCR signaling in mesoCAR T cells. A (left), equal numbers of mesoCAR- and mesoCAR-RIAD–expressing humanT cells were examined at baseline or exposed to immobilized CD3 and CD28 antibodies for 20 minutes. Lysates were prepared, equal amounts of protein wererun on an SDS gel, and then immunoblotted for phospho-ERK (pERK), phospho-Lck at tyrosine-394 (pLck-Y394), and phospho-Akt (pAkt), along with theirrespective loading controls, including actin. Right, equal numbers of humanmesoCAR- andmesoCAR-RIAD–expressingT cellswere examined at baseline or exposedto mesothelin protein immobilized on beads for 20 minutes. Lysates were prepared, equal amounts of protein were run on an SDS gel, and then immunoblottedfor phospho-ERK and actin as a loading control. B, equal numbers of murine mesoCAR- and mesoCAR-RIAD-expressing T cells were examined at baseline orexposed to immobilized CD3 and CD28 antibodies for 20 minutes. Lysates were prepared, equal amounts of protein were run on an SDS gel, and thenimmunoblotted for phospho-Akt (pAkt) or phospho-ERK. C, lysates from murine mesoCAR- and mesoCAR-RIAD–expressing T cells at baseline wereimmunoblotted for phospho-Csk at serine-364 (pCsk-S364) and pLck at tyrosine-505 (pLck-Y505). Total Erk or actin were used as loading controls (left, humanT cells; right, murine T cells).
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To evaluate the efficacy of RIAD in another CAR system, weutilized a construct that targets fibroblast activation protein(FAP) present on stromal cells surrounding the tumor (36). Weinoculated C57BL/6 wild-type mice with the pancreatic cell linePDA4662 (13). PDA4662-bearing mice were subsequentlytreated with a single dose of murine 107 FAPCAR or FAP-CAR-RIAD T cells when the tumor burden was approximately200 mm3. At this dosage of T cells, 14 days after adoptivetransfer, only FAPCAR-RIAD T cells showed statistically sig-nificant antitumor activity in this model (Fig. 3C).
MesoCAR-RIAD increased the numbers and activity oftumor-infiltrating T cells
To better understand the in vivomechanisms of the enhancedantitumor effects observed with mesoCAR-RIAD T cells,EMmeso-bearing mice were sacrificed 32 days after T-celladministration, and their tumors were pooled and processedas described in Materials and Methods. The percentage ofhuman CD3þ, CD8þ, and CD4þ cells (from total live tumorcells) was significantly higher (P < 0.05) within the tumors ofmesoCAR-RIAD–treated mice compared with mesoCAR-treated
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Figure 3.Enhancement of tumor control by mesoCAR-RIAD T cells in vivo. A, EMmeso human tumor cells (2 � 106 cells) were injected subcutaneously into the flanksof immunodeficient NSG mice and after tumors were established (�150–200 mm3 in volume), 10 million mesoCAR- or mesoCAR-RIAD–expressing humanT cells were administered via tail-vein injections. Tumor development was monitored using calipers for the next 32 days after T-cell administration, at which timeanimals were sacrificed for ex vivo analyses. B, AE17meso murine tumor cells (2 � 106 cells) were injected subcutaneously into the flanks of C57BL/6 mice,and after they were established (�150 mm3 in volume), mesoCAR- or mesoCAR-RIAD–expressing murine T cells (107) were administered via tail-veininjections. Tumors were measured, and mice were sacrificed 10 days after T-cell administration. C, PDA4662 murine pancreatic cancer cells (106 cells) weresubcutaneously injected into C57BL/6 mice, and FAPCAR- and FAPCAR-RIAD–expressing T cells (107 cells) were injected via the tail vein. Mice weremonitored and sacrificed at day 23 after T-cell administration. Statistical analyses were performed using one-way ANOVA comparing mesoCAR and mesoCARRIAD tumors at the final time point. � , P � 0.05; �� , P � 0.01; ��� , P � 0.001; ���� , P � 0.0001. At least three independent experimental replicates wereperformed. Data represent means � SEM; n ¼ 5–7 mice per group.
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Cancer Immunol Res; 4(6) June 2016 Cancer Immunology Research546
mice (Fig. 4A; Supplementary Fig. S8A). The majority of these Tcells were CD8þ, with only a small percentage being CD4þ cells(an approximately 10:1 ratio of CD8þ to CD4þ T cells).
To examine a model in which T cells can be measured at anearlier time point, we used the murine AE17meso model. Threedays after adoptive transfer, we analyzed the murine TIL popu-lation by flow cytometry. As with the human model, we alsoobserved a greater influx of murine mesoCAR-RIAD CD8þ T cellsinto the tumors by flow cytometry (P � 0.05; Fig. 4B). Again,CD8þ TILs outnumbered CD4þ TILs at an approximately 10:1ratio (Supplementary Fig. S8B).
To study the function of the adoptively transferred CARsfrom EMmeso tumors, we isolated human T cells and measuredthe ex vivo ability of these TILs ("at harvest") to kill tumor cellsand secrete IFNg compared with CAR TILs that had beenallowed to "rest" overnight (Fig. 4C). In agreement with ourprevious studies (10), freshly harvested mesoCAR TILs werehypofunctional with respect to both killing and IFNg secretionand had some recovery after "rest." In contrast, the freshlyharvested mesoCAR-RIAD TILs retained almost full cytolyticcapability and enhanced IFNg secretion compared with meso-CAR TILs.
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Figure 4.RIAD transgene enhances mesoCAR T-cell persistence and activity in vivo. A, EMmeso tumors harvested at day 32 after T-cell transfer were digested, pooled, andanalyzed using flow cytometry analysis. Cells were stained with anti-human CD8, CD3, and a live/dead stain. Left, representative flow tracings with increasedCD8 T cells in the mesoCAR-RIAD-treated tumors. Right, bar chart shows quantification of tumor digests from 7 mice per group. The frequency of liveCD4þ T cells was calculated from the difference between the total frequency of live CD3þ and CD8þ T cells. There were statistically significant increases in CD3þ,CD8þ, and CD4þ T cells in the mesoCAR-RIAD–treated tumors, although the number of CD4þ T cells was low. B, AE17meso tumors harvested at day 3 after T-celltransfer were digested and analyzed. Total tumor digests were stained with anti-murine CD45.1 to recognize adoptively transferred cells (left). Bar chart showsfrequencies by treatment group (representative of 5 mice per group). C, TILs were freshly isolated from human mesoCAR- and mesoCAR-RIAD–treated tumors(from A) or allowed to rest overnight in media. The cells were subjected to an overnight luciferase-based cytolysis assay against EMmeso cells to determinethe percentage of killing of tumor cells (left). Supernatants from the assay were also measured for IFNg production (right). Freshly harvested mesoCAR TILswere hypofunctional with respect to both killing and IFNg secretion and had some recovery after "rest." In contrast, the freshly harvested mesoCAR-RIAD TILsretained almost full cytolytic capability and enhanced IFNg secretion compared with mesoCAR TILs.
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MesoCAR-RIAD cells show enhanced migratory abilityBecause one explanation for the increased numbers of meso-
CAR-RIAD TILs was enhanced migration to tumor sites, weperformed in vitro transmigration assays in which transduced Tcellsmarked byGFPormCherry (see Supplementary Fig. S1)wereallowed to traffic toward the T-cell chemokine CXCL10. Weobserved a higher CXCL10-mediated migration rate in meso-CAR-RIAD T cells compared with mesoCAR T cells in a dose-dependent fashion in both human andmouse T cells (Fig. 5A). Tofurther explore this augmentation of chemotactic ability, weassessed the expression of the key chemokine receptor responsiblefor CXCL10 transmigration (that is, CXCR3). As shown in Fig. 5B,both human and mouse mesoCAR-RIAD cells expressed signifi-cantly (P � 0.05) higher CXCR3 than did mesoCAR T cells. Wealso studied the ability of these T cells to adhere to relevantsubstrates (fibronectin, VCAM-1, and ICAM-1) that might be
expressed on tumor endothelium or tumor cells. Overnightadhesion assays showed that both human and murine meso-CAR-RIAD cells had significantly better adhesion to these sub-strates than mesoCAR T cells (Fig. 5C). At baseline, mesoCAR Tcells did not show enhanced expression of CD11a (LFA-1) orCD18. However, mesoCAR-RIAD T cells did show significantlyincreased expression of the subunits of the VLA4 receptor (CD49aand CD29) that bind VCAM-1 and fibronectin (Fig. 5D).
DiscussionAlthough adoptive T-cell therapy using CARs for cancer has
shown great promise in the treatment of bloodborne malignan-cies (1–3), their application in the treatment in solid tumors hasnot yet been as successful. One reason for this is the inefficiency oftrafficking ofCARs into solid tumors. A second cause appears to be
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Figure 5.RIAD transgene enhances mesoCAR T-cell chemotaxis and adhesion in vitro. A, equal numbers of transduced human (left) and murine (right) mesoCAR ormesoCAR-RIAD T cells were placed in Transwell inserts and were allowed to migrate toward the indicated stimulants placed in the bottom well. After 4 hours,migrated cells were collected and counted. Data represent means � SEM; n ¼ 3 replicates per condition. B, the CAR T cells used for the transmigration assay inA were stained for baseline surface expression of CXCR3. C, overnight adhesion assays to the indicated substrates were performed using human T cells (left)andmurine T cells (right) overnight. The number of adherent cells is plotted. Data represent means� SEM; n¼ 3 replicates per condition. D, the CAR T cells used forthe overnight adhesion assay in Cwere stained for baseline surface expression of adhesion receptors. Statistical analyseswere performed using one-way ANOVA asindicated. � , P � 0.05; ��, P � 0.01; ���, P � 0.001; ���� P � 0.0001. At least three independent experimental replicates were performed.
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Cancer Immunol Res; 4(6) June 2016 Cancer Immunology Research548
the rapid loss of secretory and cytolytic function of the TILsinduced by factors within the TME (such as TGFb) and by theupregulation of intrinsic negative regulators [such as PD-1 anddiacylglycerol kinase (DGK); refs. 14, 35), a phenomenon alsodocumented in endogenous TILs (4, 5). Attempts to overcomethese limitations include introducing chemokine receptors intoCAR cells (39), reducing expression levels of DGK (35), or usingdominant-negative TGFb receptors to prevent TGFb-mediatedinactivation of cytotoxic T lymphocytes in the TME (40–42).
Soluble mediators also likely play a role. Two major inhibitorsof T-cell function present to varying degrees in the TME areadenosine and PGE2 (reviewed in refs. 43, 44). Extracellularadenosine produced by tumors cells and regulatory T cells maybe an especially prominent trigger of cAMP generation in hypoxicTMEs (reviewed in ref. 15). In this study, we demonstrated theimportance of adenosine and PGE2 in CAR dysfunction by show-ing that thepresence of either agent during coculture ofCART cellswith tumor cells results in a decrease in tumor cell killing. We alsoknow that high concentrations of PGE2 are present in our exper-imental tumor models.
The peptides RISR and RIAD (31, 33) can block PKA activity atthe TCR level in transgenic mice, suggesting that the coexpressionof these transgenes with our CAR constructs in T cells mightprotect them from cAMP/PKA-mediated immunosuppressionand thus render them resistant to both adenosine- and PGE2-mediated inhibition.
This strategy appeared highly effective. Consistent with ourproposed mechanism, we saw a decrease in the baseline phos-phorylation of Csk at S364 and reduced phosphorylation of Lck atY505 in the CAR-RIAD cells. Both human and mouse T cellsexpressing the CAR-RIAD constructs were almost completelyresistant to the in vitro inhibitory effects of adenosine and PGE2.
In addition to their resistance to adenosine and PGE2-mediatedinhibition, mesoCAR-RIAD T cells were more tumoricidal andreleased more IFNg , even in the absence of added adenosine orPGE2. This may be due to the endogenous secretion of thesemediators by the tumor cells in culture. However, the basalactivation state of mesoCAR-RIAD T cells, especially the humancells, was increased in the absence of tumor cells. In addition,exposure of cells tomesothelin-coated beads resulted in increasedcytokine secretion in themesoCAR-RIAD cells compared with themesoCAR cells. Because these studies were done in a tumor-freesetting, these data are consistent with the concept that a certainlevel of intrinsic "tonic" PKA activity exists in our CAR T cells thatmay function to set a basal signaling tone. It is likely that this"tone" is set via the effect of PKA on Csk, as the observation thatCsk activity levels establish the TCR threshold is well established(21, 45). Furthermore, a basal level of PKAactivity exists inhumanperipheral blood T cells, especially in CD8þCD45ROþ cells, thephenotype of our humanCAR T cells (46). It is unclear at this timewhether this basal activity is cell intrinsic or if there is someendogenous secretion of PGE2 or adenosine that feeds back intothe cells through cognate receptors.
In agreement with our in vitro data,mesoCAR-RIAD T cells weremore efficacious in a number of tumor models compared withmesoCAR T cells, including immunodeficient NSG mice bearingEMmeso human mesothelioma tumors treated with humanT cells, wild-type C57BL/6 mice bearing established AE17mesomouse mesothelioma tumors treated with mouse T cells, andwild-type C57BL/6 mice bearing established PDA4662 mousepancreatic tumors treated with mouse T cells. Most of this effect
was probably due to resistance to the inhibitory influences ofPGE2 and/or adenosine in the TME, because freshly isolatedhuman mesoCAR-RIAD TILs had much more ex vivo antitumoractivity than did similarly processed CAR TILs. However, it ispossible that the increased basal and stimulated activity of themesoCAR-RIAD T cells may also be playing a role. Despite theirenhanced reactivity, no nonspecific activity against non-antigen–expressing cells in vitro or in vivo was detected.
Using these models, at least two mechanisms responsible forthe enhanced efficacy of mesoCAR-RIAD T cells were identified.Consistent with our in vitro data, mesoCAR-RIAD TILs were morefunctional than mesoCAR TILs. As previously reported (10),freshly isolated human TILs from mesoCAR T cell–treated micehad reduced ability to kill tumor cells or release IFNg in our ex vivoassay. In contrast, freshly isolated TILs from mesoCAR-RIAD Tcell–treatedmicewere nearly equivalent in function to the infusedproduct.
We also observed (somewhat unexpectedly) that the number ofCD8 mesoCAR T cells infiltrating the tumors was significantlyhigher in the mesoCAR-RIAD T cell–treated mice compared withthe mesoCAR T cell–treated mice in both the NSG and syngeneicmodels (Fig. 4A and B). Although both CD4þ and CD8þ T cellsincreased, the CD8þ cells outnumbered the CD4þ by a ratio of10:1. We did not directly study the relative importance of CD8þ
versus CD4þ T cells in our model, but there is good evidence thatboth types of cells are needed for full CAR T-cell efficacy (47, 48).In the NSG model, this could be due to enhanced persistenceand/or increased trafficking of mesoCAR-RIAD T cells. However,seeing increased infiltration of adoptively transferred T cells intumors in the syngeneic mouse model at only 3 days afteradoptive transfer suggests that the increased infiltration was dueto enhanced trafficking. Transmigration assays using the chemo-kineCXCL10 (whichbinds to theCXCR3 chemokine receptor thatis highly expressed on CAR T cells) revealed significantly bettermesoCAR-RIAD T-cell migration. Overnight adhesion assays alsoshowed that mesoCAR-RIAD T cells showed significantly betteradhesion to fibronectin, ICAM, and VCAM ligands, thus poten-tially contributing to their enhanced tumor trafficking. Thesechanges were associated with increased expression of the chemo-kine receptor CXCR3 and the CD49d integrin (VLA-4). Thedifferential baseline expression of these adhesion receptors isreflective of the complexity of leukocyte mobilization during animmune response, as previously reported (16).
There is abundant literature describing the effects of PKA onleukocyte cell migration and adhesion, although the reportedeffects are complex and somewhat cell type specific (reviewedin ref. 17). With regard to lymphocytes, elevated intracellularcAMP inhibits RhoA activation and integrin-dependent leuko-cyte adhesion induced by chemoattractants (49). This obser-vation fits with our data showing that inhibition of PKA byRIAD augmented T-cell migration and adhesion in vitro andT-cell migration in vivo.
Our studyhas a number of potential limitations. In ourmodels,PGE2 concentrations were relatively high, but different tumorslikely vary in their expression of PGE2 or adenosine, which couldaffect the degree of RIAD-induced augmentation seen. The poten-tial immunogenicity of the RISR–RIAD peptide may also be anissue for clinical translation; however, analysis of RISR/RIADin silico showed no immunogenic neoepitopes for common HLAtypes. Lastly, the intrinsic limitations in the animal models usedshould be considered because no animal model can completely
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predict the response that might occur in patients. The syngeneicmouse models using mouse CAR T cells have an intact immunesystem and complete species compatibility, but the behaviorand persistence of adoptively transferred mouse T cells areclearly different than those of human T cells. The NSG modelwe used allows the study of human T cells and human tumors,but lacks many elements of an actual human TME (i.e., regu-latory T cells, tumor-associated macrophages, etc.), and is achimeric system in which not all mouse cytokines are cross-reactive with human cells and vice versa. However, by showingsimilar effects using both types of models (with humanand mouse T cells) and different CARs and CAR targets, wefeel that the potential applicability of our approach to humanCAR T-cell clinical trials has been shown.
There is a clear need for approaches to overcome the manypotential limitations ofCARs in the treatment of solid tumors. Thegenetic addition of the RIAD transgene (which is rather small)should be feasible for virtually any CAR (or T cells with transgenicTCRs) in a bicistronic fashion. We thus propose that the additionof RIAD could be an important strategy to augment CAR efficacyin the treatment of solid tumors where an immunosuppressivemilieu exists, trafficking is limited, and antitumor responses havebeen suboptimal.
Disclosure of Potential Conflicts of InterestS.M. Albelda reports receiving commercial research support from Novartis.
No other potential conflicts of interest were disclosed by the other authors.
Authors' ContributionsConception and design: K. Newick, E. Pur�e, S.M. AlbeldaDevelopment of methodology: K. Newick, V. Kapoor, S.M. AlbeldaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Newick, S. O'Brien, A. Lo, E. Pur�e, S.M. AlbeldaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Newick, S. O'Brien, E. Pur�e, S.M. AlbeldaWriting, review, and/or revision of the manuscript: K. Newick, S. O'Brien,E. Pur�e, S.M. AlbeldaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Newick, S. Maceyko, A. Lo, S.M. AlbeldaStudy supervision: S.M. Albelda
AcknowledgmentsThe authors thank Dr. Omkar Kawalekar for providing mesothelin-coated
beads and Dr. Astero Klabatsa, Soyeon Kim, and Naomi Saint Jean for helpfulinput and discussion.
Grant SupportThis project was supported by NCI grants P01 CA 66726-07 (to S.M. Albelda),
R21 CA169741, R01 CA141144 and R01 CA 172921 (to S.M. Albelda andE.Pur�e),a researchgrant fromNovartisPharmaceuticalsCorporation,and fundingfrom the LungCancer Translational Center of Excellence atUPenn. K.Newickwasfunded by T32 HL07586. S. O'Brien was funded by T32 CA09140.
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 October 23, 2015; revised February 18, 2016; accepted March 4,2016; published OnlineFirst April 4, 2016.
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2016;4:541-551. Published OnlineFirst April 4, 2016.Cancer Immunol Res Kheng Newick, Shaun O'Brien, Jing Sun, et al. Blocking Protein Kinase A LocalizationAugmentation of CAR T-cell Trafficking and Antitumor Efficacy by
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