T-Cell Receptor Stimulation Enhances the ......Nov 08, 2019 · We therefore tested the efﬁcacy of CD19.CAR-VSTs in eight HSCT recipients in remission from high-risk, CD19þ B-cell

Clinical Trials: Immunotherapy

T-Cell Receptor Stimulation Enhances theExpansion andFunction of CD19Chimeric AntigenReceptor–Expressing T CellsNatalia Lapteva1,2, Margaret Gilbert1, Iulia Diaconu1, Lisa A. Rollins1, Mina Al-Sabbagh1,Swati Naik1,3,4, Robert A. Krance1,3,4, Tamara Tripic1, Manasa Hiregange1,Darshana Raghavan1, Olga Dakhova1, Rayne H. Rouce1,3,4, Hao Liu1,5, Bilal Omer1,3,4,Barbara Savoldo1,3, Gianpietro Dotti1,2,6, Conrad Russel Cruz1, Keli Sharpe1, Melissa Gates1,Aaron Orozco1, April Durett1, Elizabeth Pacheco1, Adrian P. Gee1,3, Carlos A. Ramos1,6,7,Helen E. Heslop1,3,4,6,7, Malcolm K. Brenner1,3,4,6,7, and Cliona M. Rooney1,2,3,4,8,9

Abstract

Purpose: Current protocols for CD19 chimeric antigenreceptor–expressing T cells (CD19.CAR-T cells) require reci-pients to tolerate preinfusion cytoreductive chemotherapy,and the presence of sufficient target antigen on normal ormalignant B cells.

Patients and Methods:We investigated whether additionalstimulation of CD19.CAR-T cells through their native recep-tors can substitute for cytoreductive chemotherapy, inducingexpansion and functional persistence of CD19.CAR-T even inpatients in remissionof B-cell acute lymphocytic leukemia.Weinfused a low dose of CD19.CAR-modified virus-specific T

cells (CD19.CAR-VST) without prior cytoreductive chemo-therapy into 8 patients after allogeneic stem cell transplant.

Results: Absent virus reactivation, we saw no CD19.CAR-VST expansion. In contrast, in patients with viral reacti-vation, up to 30,000-fold expansion of CD19.CAR-VSTs wasobserved, with depletion of CD19þ B cells. Five patientsremain in remission at 42–60þ months.

Conclusion: Dual T-cell receptor and CAR stimulation canthus potentiate effector cell expansion and CAR-target cellkilling, even when infusing low numbers of effector cellswithout cytoreduction.

IntroductionChimeric antigen receptor–expressing T cells directed to the

CD19 antigen (CD19.CAR-T) have proved remarkably effectivein treatment of pre-B acute lymphocytic leukemia (ALL) andother B-cell malignancies (1–3). In principle, signaling throughCD19 and other CARs could be reinforced by concomitant orsequential signaling through the native T-cell receptor (TCR).Demonstration of such an effect would have two major benefits,by inducing CAR-T–cell expansion and reducing the toxicity of

lymphodepleting chemotherapy. Optimal CAR-T–cell expansionand persistence currently requires administration of toxic cytor-eductive chemotherapy, whichmay be undesirable or impracticalfor some patients (4). Even when cytoreductive therapy is anacceptable option, engraftment of CAR-T cells may be insufficientfor sustained antitumor activity if there are limiting numbers ofnormal and malignant target cells expressing the CAR-targetantigen (5). We therefore examined whether combined TCR andCAR stimulation can obviate preinfusion cytoreduction, andwhether it can also expand functional CAR-T cells even whenthese cells are infused in small numbers and/or their cognateantigen is present at insufficient levels for their desired expansionand persistence.

To assess theputative benefits of dual TCR/CAR stimulation,wechose patients who received allogeneic hematopoietic stem celltransplantation (HSCT) as treatment for high risk pre-B-cellleukemia. These patients are at risk both for leukemic relapseand for severe viral infections, so that a T-cell product with bothantileukemic and antiviral activity could provide a safe means toprotect patients from both. Adoptively transferred virus-specific Tcells (VST) proliferate extensively after infusion into recipients ofT-cell–depleted allogeneic HSCT with viral reactivation, and thenreturn to the long-termmemory population, where they retain theability to reexpand in response to virus reactivation (6). Wereasoned that if donor VSTs were modified with leukemia-specific CD19.CAR, they also should expand in the presence ofviral infection or reactivation and protect patients from both viralinfections and leukemic relapse, thereby achieving dual endsthrough a single means.

1Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Meth-odist Hospital, Texas Children's Hospital, Houston, Texas. 2Division of Immu-nology, Department of Pathology, Baylor College of Medicine, Houston, Texas.3Division of Hematology and Oncology, Department of Pediatrics, BaylorCollege ofMedicine, Houston, Texas. 4Texas Children's Hospital, Houston, Texas.5Division of Biostatistics, Dan L. Duncan Cancer Center, Baylor College ofMedicine, Houston, Texas. 6Department of Medicine, Baylor College of Medicine,Houston, Texas. 7Houston Methodist Hospital, Houston, Texas. 8Program ofTranslational Biology and Molecular Medicine, Baylor College of Medicine,Houston, Texas. 9Department of Molecular Virology and Microbiology of BaylorCollege of Medicine, Houston, Texas.

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

Corresponding Author: Cliona M. Rooney, Baylor College of Medicine, OneBaylor Plaza, Houston, TX 77030. Phone: 832-824-4693; Fax: 832-825-4732;E-mail: [email protected]

Clin Cancer Res 2019;XX:XX–XX

doi: 10.1158/1078-0432.CCR-18-3199

�2019 American Association for Cancer Research.

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We therefore tested the efficacy of CD19.CAR-VSTs ineight HSCT recipients in remission from high-risk, CD19þ B-cellALL (B-ALL). Patients received donor T cells specific for cytomeg-alovirus (CMV), Epstein–Barr virus (EBV), and adenovirus(multivirus-specific T cells) modified with a second generationCD19.CAR. CD19.CAR-VSTs could be detected by PCR for amedian of 182 weeks (range 8 weeks to 5 years), but only in thepresence of viral reactivation was there a substantive expansionof CD19.CAR-VSTs and associated B-cell aplasia, despite thepresence of significant numbers of normal B cells in all patientsat the time of infusion. Hence, concomitant signaling through theTCR and CAR can enhance the proliferation and the function ofCAR-VSTs.

Patients and MethodsPatients

The studywas conducted in accordance with the U.S. CommonRule following approval by an Institutional Review Board ofBaylor College of Medicine the Recombinant DNA AdvisoryCommittee and the FDA. Written consent was obtained from allstudy subjects.

All patients were in remission from B-ALL and had nodetectable disease at the time of infusion (MRD-negative). Thesubject's stem cell donor was also the donor for the CAR-VSTproduct that was generated from up to 100 mL of peripheralblood (Fig. 1A). Patients were infused at least 1 month fromlymphodepleting antibodies and off steroids for treatment ofGVHD at the time of infusion. They were allowed to remain onother GVHD prophylaxis. Patients were eligible to receive asingle dose of 1 � 107 CD19.CAR-VSTs/m2 after day 30 postHSCT and were subsequently monitored for toxicity and detec-tion of transduced VSTs, virus-specific immunity, and B-cellimmune reconstitution.

Seven of 8 patients received myeloablative conditioning ther-apy and 1 received reduced intensity conditioning therapy. Fourpatients received transplants from matched related donors, 2from matched unrelated donors, and 2 from mismatched unre-lated donors. Two of the 8 patients in this cohort had received aprior myeloablative allogeneic HSCT. Donors were procured for

cell manufacture at median of 11 days prior to transplant (range1–31 days prior to HSCT).

Adenoviral and retroviral vectorsBoth vectorswere produced by the Vector Production Facility of

the Center for Cell andGene Therapy, Baylor College ofMedicine.TheCD19.CAR scFvdomain, FMC-63, targeting theCD19antigenwas provided by Heddy Zola (Child Health Research Institute,Women's and Children's Hospital, Adelaide, South Australia,Australia; ref. 7). The CAR.CD19-28z vector was generated asdescribed previously (8–10). Briefly, a spacer region derived fromthe human IgG1-CH2CH3 domain was cloned in-frame betweenthe scFv and the signaling domains and cloned into the SFGretroviral backbone. Clinical grade packaging cell lines weregenerated with the use of PG13 cells (gibbon ape leukemia viruspseudotyping packaging cell line; CRL-10686, ATCC).

To generate antigen-presenting cells (APC) for VST generation,we used a previously described adenoviral vector, Ad5f35-pp65containing the immunodominant pp65 protein of CMV (11, 12),to transduce EBV-transformed B lymphoblastoid cell lines (LCL).Ad5f35-pp65–transduced LCLs presented de novo–expressedpp65, the hexon and penton proteins from the Ad vector andendogenous EBV proteins (13).

Generation of EBV-transformed B LCLsLCLs were generated in our Good Manufacturing Practices

(GMP) facility by infection of peripheral blood mononuclearcells (PBMC) from each stem cell donor, with a clinical grade EBVproduced by a clinical grade B95-8 producer cell line, in thepresence of cyclosporin A (1 mg/mL; Sandoz) as described pre-viously (14). Autologous LCLs were irradiated and used as APCsafter transduction with Ad5f35-pp65.

Generation of multivirus-specific T cellsVSTs specific for CMV, EBV, and adenovirus were expanded

from 40–60 mL of peripheral blood from the stem cell donor.PBMCswere purifiedusing Lymphoprep (Axel-Shield PoCAs). Togenerate APCs for the first stimulation of VSTs, donor LCLs weretransduced with Ad5f35-pp65 at 10,000 virus particles per cell,irradiated, and cocultured with autologous PBMCs at a 1:40 ratioof LCL:PBMCs in T-cell medium comprising 45% advancedRPMI1640 (Hyclone), 45% Click medium (Irvine Scientific),2 mmol/L/L GlutaMAX TM-I (Invitrogen), and 10% FBS(HyClone, Thermo Fisher Scientific) in presence of 10 ng/mL IL7and 100ng/mL IL15 (R&DSystems). In this protocolwemodifiedthe manufacturing strategy from that reported previously (15)to ensure early transduction of central memory CMV, EBV,and adenovirus-specific T lymphocytes (VST; ref. 16). Threedays after culture initiation, VSTs were transduced with theg-retroviral vector expressing CD19.CAR-28�z. Transduced VSTs(CD19.CAR-VSTs) were cultured for 8–10 dayswith IL7 and IL15,and if sufficient cells were obtained, they were cryopreserved forinfusion. Otherwise, CD19.CAR-VSTs were restimulated withautologous Ad-pp65-LCLs for another 8 days. Ten products weremanufactured and eight were infused. For these 10 cell lines,viability by 7-AAD staining was 92% � 4%, CD3þ 88% � 10%,CD3þCD4þ 15%� 8%, CD3þCD8þ 63%� 20%, CD56þCD3neg

[natural killer (NK) cells] 11%� 10%, CD4þCD45RAnegCD62Lþ

10.7%�4.2%, andCD8þCD45RAnegCD62Lþ 30.1%� 21%. Theaverage CD19.CARþ frequency within the CD19.CAR-VSTs was63%�20%.Ofnote, themeanfluorescent intensity ofCARþVSTs

Translational Relevance

It is widely accepted that cytotoxic lymphodepletion priorto adoptive T-cell immunotherapy is essential for the expan-sion and antitumor efficacy of adoptively transferred chimericantigen receptor–modified T cells (CAR-T cell). However, notall patients, in particular hematopoietic stem cell transplant(HSCT) recipients, can tolerate this treatment. Here we showthat viruses can induce exponential expansion of CD19chimeric antigen receptor expressing virus-specific T cellswithout cytokine release syndrome or neurotoxicity in HSCTrecipients at high risk for relapse of B-cell acute lymphoblasticleukemia. This work provides proof of the concept that CAR-Tcells can be expanded via their T-cell receptor (TCR) withoutlymphodepletion and supports investigation of viral vaccinesor oncolytic viruses to expandT cells bispecific for cognate viralantigens via their TCR and for tumor antigens via their CAR.

Lapteva et al.

Clin Cancer Res; 2019 Clinical Cancer ResearchOF2




did not differ from that of similarly transduced T cells stimulatedwithCD3andCD28antibodies (Supplementary Fig. S1). All T celllines generated for infusion were generated in the GMP facilityand tested negative for Mycoplasma and bacteria.

Cytotoxicity assaysCytotoxic activity of CD19.CAR-VSTs was measured using a

standard 6-hour Cr51 (Perkin Elmer) release assay. Percent-specific lysis was calculated as specific lysis [(experimentalrelease � spontaneous release)/(maximum release � spontane-ous release)] � 100. To assess the CAR function, we tested theability of each line to kill CD19þ Raji Burkitt lymphoma cells,while donor-derived LCLs provided additional CD19þ and EBVþ

autologous target cells (Supplementary Fig. S2A). Raji cells wereauthenticated in July 2017 using short tandem repeat profilesmethod by MD Anderson Cancer Center Characterized Cell Linecore (Houston, TX) and Mycoplasma tested using MycoAlert(Lonza) on a monthly laboratory schedule ofMycoplasma testing.Target tumor cells used in the assays were cultured for no longerthan 6 months. Less than 10% lysis of recipient or donor phy-

tohaemagglutinin-activated T-cell blasts in chromium releaseassays was a product release criterion.

To test NK-cell cytotoxicity, NK cells from patient blood wereexpanded using a previously reported protocol (17). Target cellswere chromium51-labeled donor-derived CD19.CAR-VSTs, GD2.CAR-transduced VST (control), nontransduced VSTs (NT), orK562 cells.

IFNg enzyme-linked immunospot assayIFNg enzyme-linked immunospot (ELISpot) assays were used

to test the frequency of T cells specific for each virus, both in theinfusion product, and in patients' PBMCs preinfusion andon weeks 1, 2, 4, 8, 12, and months 6 and 12 after infusion.CD19.CAR-VSTs (1 � 105/well; Supplementary Fig. S2B) orPBMCs (3 � 105/well) were plated either with 1 � 105 LCLs orwith 1 mg/mL of overlapping peptide libraries (pepmixes) span-ning EBNA-1, LMP1, LMP2, BARF-1 of EBV, pp65 of CMV, andhexon and penton of adenovirus (JPT Peptide Technologies).Data are presented with background (no stimulation) subtracted.In some assays, HLA-negative K562cs (18) cells expressing

B-ALL pa�ents in remission a�er allo-HSCT A

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Figure 1.

Outline of study design and CD19.CAR-VST cell expansion. A, Stem cell donor blood, obtained between 1 and 31 days before HSCT, was used to manufactureCD19.CAR-VSTs. Cells were infused into patients with B-ALL in remission of HSCT between day 74 and 158 after HSCT. Immune cell reconstitution was monitoredby flow cytometry, and virus specificity was analyzed by INFg ELISpot at the indicated times after infusion. The presence of the CD19.CAR transgene wasanalyzed using qPCR assay. B, Expansion of CD19.CAR-VSTs in patients with EBV virus load. Relatively low levels of expansion of CD19.CAR-VSTs were detectedby qPCR in 5 patients without detectable EBV, CMV, or adenovirus (black dashed lines, individual patients; black solid line, mean values). CD19.CAR-VSTsexpanded in 3 patients with reactivated EBV (red dashed lines, each patient; red solid line, mean values). C, No significant expansion of CD19.CAR-VSTs in theblood of representative patient with no detectable viral reactivation/infections.

TCR Stimulation for Enhanced CAR-T–Cell Function

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costimulatory molecules were added at 1 � 105 cells/well. Theassay was developed as described previously (6, 19).

Transgene qPCRTo measure the frequency of circulating, transduced CD19.

CAR-VSTs, the CD19.CAR transgene copy number was analyzedin patient PBMC using qPCR as described before (15). Briefly,genomic DNA was extracted from patient PBMCs and analyzedwith primers fromApplied Biosystems (Thermo Fisher Scientific).Forward primer 50-CCACCCGCAAGCATTACC-30, reverse primer50-CGCTCCTGCTGAACTTCACTCT-30 andprobe FAM-CACCAC-GCGACTTCGCAGCCT-30 TAMRA. Primers were used at a finalconcentration of 500 nmol/L each, and the probe was used ata final concentration of 120 nmol/L. qPCR was performedwith TaqMan Universal Master Mix (Applied Biosystems) on7900H using default TaqMan program: 50�C, 2 minutes; 95�C,10 minutes, (95�C, 15 seconds; 60�C, 1 minute) � 40 cycles.

Coculture of CD19.CAR-VSTs with B cellsB cells were purified by negative selection using a human BCell

Isolation Kit (Stemcell Technologies). CD19.CAR-modifiedEBV-specific T cells (EBVST) were cocultured with B cells at a1:1 ratio, with or without overlapping peptide libraries spanningthe protein sequences (pepmixes) of EBNA-1, LMP-1, LMP-2, andBARF-1. Cells were stained with anti-CD3 and goat anti-humanIgG (HþL) AF647 antibodies and acquired for 100 seconds usingGallios FlowCytometer (BeckmanCoulter Inc.) on day 0 and 7 ofeach stimulation.

Flow cytometryCD19.CAR expression levels in VSTs and PBMCswasmeasured

by flow cytometry using goat anti-human-IgG (HþL)-AF647antibody (Jackson ImmunoResearch) and analyzed using aFACSCalibur flow cytometer. VSTs were phenotyped usingfluorochrome-conjugated CD3, CD4, CD8, CD16, CD19,CD45RA, CD45RO, CD62L, CCR7, CD27, and CD28 mAbs (BDBiosciences) and acquired using BD FACSCanto-II (3-laser)Cytometer and analyzed with FACSDiva Software v6.1.3.CD19.CAR expression was not analyzed in follow-up sampleswith low transgene copy numbers by qPCR.

Detection of EBV and adenovirus DNA in PBMCs by qPCRDNA was isolated from purified PBMCs using Blood DNA

Isolation Kit (Qiagen). A total of 500ng of DNA was analyzedby qPCR as described previously (20). Adenovirus andCMVDNAwere measured by Viracor IBT Laboratories.

Genotyping of Fcg receptorsGenotyping of FcgRIIA, FcgRIIIA, and FcgRIIIB was conducted

on genomic DNA extracted from patient PBMCs (QIAamp DNABlood Kit, Qiagen) by PCR using Platinum Taq DNA Polymerase(Invitrogen). Primers and PCR conditions adapted from previ-ously published methods (21–23).

Statistical analysisData are presented as mean � SD and were analyzed using

paired two-tailed Student t test to determine statistical signifi-cance (P < 0.05) of differences when comparing two treatmentgroups. The association of CD19.CAR-VST expansion and EBVreactivation was analyzed using Fisher exact test. Data wereanalyzed using GraphPad Prism version 5.0 Software (GraphPadSoftware Inc).

ResultsPatient characteristics, virus reactivation, and expansion ofCD19.CAR-VSTs

Patient demographics are shown in Table 1. Eight pediatricallogeneic HSCT recipients in remission from B-ALL received asingle dose of 1� 107 CD19.CAR-VSTs/m2 (Fig. 1A). Themedianpatient age at enrollment was 8 years (range 3–20 years) andmedian post-HSCT day of infusion was 112 days (range 74–158 days). There were no infusion-related toxicities. At the timeof infusion, absolute peripheral blood lymphocyte counts were946� 569/mL, and average CD19þ B-cell counts were 273� 258cells/mL; hence patients were not severely lymphopenic (Table 1).All patients were negative forminimal residual disease at the timeof treatment.

We measured the frequency of CD19.CAR-VSTs in peripheralblood before and after infusion by stimulating PBMCs with viralantigens in IFNg ELISpot assays, and by determining the copynumber of transgene-positive/expressing cells by PCR and flowcytometry, respectively. We determined the in vivo antiviralactivity of these cells by measuring changes in viral load by PCR.EBV DNA was detected in circulating PBMCs in only 3 patients,while no patients reactivated CMV or experienced detectableadenoviral infection. Therefore, in 5 patients, the only apparentsource of CD19.CAR-VST stimulation was the normal B cells(CAR-mediated stimulation), while in 3 patients EBV antigenswere also available for concomitant CAR- and TCR-mediatedstimulation.

In the 5 patients without detectable EBV, CMV, or adenovirusreactivation or infection, there was a relatively low level of

Table 1. Patient characteristics at the time of T-cell infusion

Subject no. Age (y)Disease statusbefore HSCT

Days afterHSCT

Lymphocytes/mLblood at infusion

CD19þ B cell# at infusion

EBV copies/mgPBMC DNA CNI

Tacrolimus level at time infusionin ng/mL (if applicable)

1 8 CR2 104 316 142 3,445 Weaning 4.32 3 CR1 158 681 176a ND No N/A3 14 CR2 89 598 453 149 Weaning 3.84 20 CR3 83 1,404 357 255 Weaning 2.25 4 CR2 74 1,851 1,038 ND Weaning <26 8 CR3 120 878 109 ND No N/A7 9 CR3 129 1,487 737 ND No N/A8 7 CR1 139 350 112 ND No N/AMean � SD 9 � 6 N/A 112 � 29 946 � 569 273 � 258 481 � 1,201

NOTE: Boldface indicates mean � SD.Abbreviations: CNI, calcineurin inhibitors; CR, complete remission #; N/A, not applicable; ND, not detected (limit of detection is 10 copies).aAnalyzed on day 6 after infusion.

Lapteva et al.





expansion of VSTs [as measured by IFNg ELISpot assays or ofCD19.CAR-VSTs by PCR (Fig. 1B) or flow cytometry (Fig. 1C)].Transgene expression in PBMCs was �20 copies/mg PBMC DNAfor most of the timepoints in all of these patients, except forpatients 2 and 6, whose transgene levels increased at week 2 to77.8 and 56.4 copies/mg PBMC DNA, respectively.

Three patients (1, 3, and 4) had measurable peripheral bloodEBV load at the time of infusion (Table 1), and this was associatedwith a correspondingly greater expansion of transgene-positiveT cells in PBMC than in patients without a detectable virus load(P ¼ 0.02; Fig. 1B).

Patient 1 had the highest virus load with 3,445 EBV gen-omes/mg PBMC DNA at the time of infusion, peaking at 5,204copies/mg by 4 days post-infusion, and decreasing to undetectablelevels by day 18 (Fig. 2A). This patient also showed the greatestexpansion of CD19.CAR-VSTs, increasing from 7.6 transgenecopies/mg at 3 hours post-infusion to 30,810 copies by week 2.

CARþ cells have persisted for over 60 months, at which time thetransgene was detected at 0.8 copies/mg DNA. In this patient,CD19.CAR-VSTs could be detected by flow cytometry at week 2post-infusion (Fig. 2B) with 32% of CD3þ T cells expressing theCAR. This striking CD19.CAR-VST cell expansion coincided withcomplete B-cell aplasia, until month 6, when B cells becamedetectable at low levels (18/mL; Fig. 2C); B lymphopenia persistedfor 17 months post-infusion. Coincident with increasing trans-gene expression, we detected an increase in the frequency of EBV-specific T cells that remain detectable for at least 2 years post-infusion (Fig. 2D). Patient 1 experienced no cytokine releasesyndrome, neurotoxicity, or other CD19.CAR-VST–associatedtoxicities even when CD19.CAR-VSTs comprised 32% of periph-eral blood CD3þ T cells (Fig. 2B). This patient remains in remis-sion from B-ALL at 60þ months.

Patient 3 had a low level of EBV reactivation (149 copies/mgDNA) and concomitant, but transient, CD19.CAR-VST

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CD19.CAR-VST expansion associated with EBV reactivation in patient 1. A, Expansion of CD19.CAR-VSTs and control of reactivated EBV. B, CD19.CAR-VSTs weredetectable by flow cytometry in patient's peripheral blood (top). CD19þ normal B cells were depleted from blood on week 2 after infusion (bottom). C, Normal Bcells were completely depleted from the blood of patient fromweek 2 until week 38. CD19þ B-cell number remains low (<100 CD19þ cells/mL) at 18 months afterinfusion. D, Reactivity in PBMCs to three viruses (EBV, CMV, and adenoviruses) was measured in PBMCs by IFNg ELISpot. EBV responses were analyzed withLCLs and overlapping peptide libraries to EBNA1, LMP1/2, BZLF1, BARF-1, and EBNA3a, 3b, and 3c (EBVpx). Responses to CMVwere analyzed using overlappingpepmixes to pp65 (CMV px); adenovirus responses were analyzed using overlapping peptide libraries for hexon and penton (Ad px). LCLs alone were plated(timepoints 9, 12, 24, and 30months) as controls and did not produce IFNg (0 SFC/well).






expansion, with the highest peak of 215 copies/mg PBMCDNA atweek 4, dropping to 6.2 copies/mgDNA atmonth 6 (Fig. 3A). Thispatient had EBV-specific T-cell reactivity throughout the study.Adenovirus-specific T cells were undetectable at the time ofinfusion, but increased in frequency at week 4 post-infusion andwere still detected 3 months after T-cell infusion, even thoughadenovirus was not detected in the patient's blood (Supplemen-tary Fig. S3A). Patient 3 did not develop CD19þ normal B-cellaplasia, relapsed at 15 months after T-cell infusion, and died ofdisease.

Patient 4 had an EBV load of 255 copies/mg peripheral bloodDNA at the time of infusion. CD19.CAR-VSTs increased from 5copies at the 3-hour timepoint to 4,426 transgene copies/mg DNAon day 11 post-infusion when they were detectable by flowcytometry (Fig. 3B and C). Although no increase in the frequencyof T cells reactive with EBV pepmixes representing EBV type 2latency antigens (LMP1, LMP2, EBNA1, and BARF1) was detectedby IFNg ELISpot (Supplementary Fig. S3B), there was an increasein the response to the autologous EBV-LCL that expressesmultipleEBV latency and lytic cycle antigens. The high EBV virus load

Figure 3.

Expansion of CD19.CAR-VSTs upon EBV reactivation in patients 3 and 4.A and B, qPCR data for the CD19.CAR and EBV in patients 3 and 4, respectively.C, CD19.CAR-VSTs were detected by flow cytometry PBMCs from patient 4 on day 10 after infusion, and CD19þ B cells were depleted coincident withCD19.CAR-VST expansion. D, Transient depletion of CD19þ B cells and expansion of CD19.CAR-VSTs in patient 4 during the study period.

Table 2. Clinical outcomes, CD19.CAR-VST cell expansion, and B-cell depletion

Patient #Highest CD19.CAR copies/mgDNA (time after infusion)

Clinical outcome(time after infusion)a

Reported MRD status afterCD19.CAR-VST therapy

1 30,810 (week 2) Disease-free (60 moþ) Negative (55 mo A.I.)2 77.8 (week 2) Relapsed 41 mo after T-cell infusion Detected MRD (41 mo A.I.)3 215 (week 4) DOD 13 mo after T-cell infusion Detected MRD (3 mo A.I.)4 4,426 (week 1.5) Disease-free (42 moþ) Negative (21þ mo A.I.)5 5.17 (month 30) Disease-free (48 moþ) Negative (42þ mo A.I.)6 56.4 (week 2) Disease-free (48 moþ) Negative (47þ mo A.I.)7 12.7 (week 1) DOD 11 mo after T-cell infusion Detected MRD (2 mo A.I.)8 14 (week 2) Disease-free (42 moþ) Negative (19þ mo A.I.)

Abbreviations: A.I., after T-cell infusion; DOD, died of disease; mo, month; MRD, minimal residual disease.aReported at the time of article submission.

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decreased to undetectable levels byweek2 after infusion (Fig. 3B).Normal CD19þ B cells were depleted in this patient for 2 months(Fig. 3D) and the patient remains in remission for more than42 months after CD19.CAR-VST infusion with transgene detect-able at 12.9 copies/mg DNA.

Of note, the higher frequency of CD19.CAR-VSTs in patients 1and 4was accompanied by normal B-cell aplasia (See Figs. 2B andC and 3D).

Overall, 5 of the 8 patients remain disease free, 2 patientsdied of disease (patients 3 and 7), and patient 2 relapsedat 41 months post T-cell infusion and was transferred to acommercial CD19.CAR-T–cell therapy (Table 2). There was noincrease in the frequency of CD19.CAR-VSTs at the time ofrelapse as measure by PCR for the transgene in peripheralblood.

Analysis of CD19.CAR-VSTs ex vivoCD19.CAR-VSTs showed little expansion in 5 of the 8 patients,

despite the high numbers of circulating B cells (SupplementaryTable S1), suggesting that while CD19.CAR-VSTs could expandrapidly in response to TCR stimulation, they expanded poorly inresponse to CAR stimulation. We investigated the mechanism for

this failure, first by determining whether CD19.CAR-VSTs couldproliferate in vitro in response to normal CD19þ B cells or EBV-antigen–expressing CD19þ B cells. CD19.CAR-VSTs coculturedwith purified autologous CD19þ B cells from three differentdonors could recognize and kill normal B cells (Fig. 4A), butproliferated poorly (total fold expansion 13 � 10-fold) inresponse to serial stimulation with autologous B cells. Howeverproliferation was enhanced when the autologous B cells werepulsed with EBV pepmixes (246 � 115-fold; Fig. 4B and C),supporting the in vivo indication that stimulation through the TCRwas more effective at inducing T-cell proliferation than stimula-tion through the CAR.

We next determinedwhether lack of CD19.CAR-VST expansionfollowing CAR stimulation alone could be explained by the Fcbinding domain of the IgG1-derivedCH2-CH3 spacer of theCAR.Hudecek and colleagues suggested that an IgG4-derivedCH2-CH3 spacer element in CARs can bind to the Fc receptoronmyeloid cells, leading to activation-induced cell death (AICD)in vivo (24). To test whether binding of our IgG1 CH2.CH3domain by monocytes could induce AICD or even direct killingof CAR-VSTs, we cocultured CD19.CAR-VSTs and NT-VSTs withCD14þ-purifiedmonocytes for 3 days. Both CD19.CAR-VSTs and

Figure 4.

CD19.CAR-VSTs were cytotoxic to autologous normal B cells and expanded in vitro. A, B cells were purified by positive selection with CD19 microbeads andcocultured at 1:1 ratio with CD19.CAR-VSTs or NT-VSTs for 3 days. CD19.CAR-VSTs eliminated B cells (CD20þ) on day 1 of culture. B, CD19.CARþ EBVST cellexpansion in response to multiple stimulations with B cells pulsed with EBV pepmixes or B cells alone. C, Expansion of CD19.CARþCD3þ EBVSTs on day 7 ofcoculture with B cells alone (13� 10-fold cumulative expansion) or B cells pulsed with EBV pepmixes (246� 115-fold). Representative results from one donorafter the third stimulation are shown.






NT-VSTs showed high viability after 3 days of coculture withmonocytes in the presence or absence of EBV pepmixes (Fig. 5A),and retained their ability to respond to EBV pepmixes (Fig. 5B).Monocytes became undetectable after 3 days of coculture witheither CD19.CAR VSTs or NT-VSTs (Fig. 5C) both in the presenceand absence EBVpepmixes. BecauseNK cells also express FcRs, weexpanded NK cells from patients 1 and 5 as effector cells andadded autologous CD19.CAR-VSTs as targets. We found nosignificant death of CD19.CAR-VSTs induced by either patient'sNK cells (Supplementary Fig. S4).

Finally, we excluded the possibility that the affinity of patientFcRs for immunoglobulin constant regions determined the in vivoexpansion and persistence of the CH2-CH3 containing CD19.CAR-VSTs, rather than the presence of TCR stimulation from viralreactivation. We analyzed known polymorphic regions of CD32and CD16 FcRs that might mediate binding and killing (21–23).

RIIA: H/H131, high affinity binding; H/R131, intermediate;R/R131, and low affinity binding to IgG; Polymorphism RIIA:V/V158, high affinity; V/F158, intermediate; and F/F158, lowaffinity binding; and polymorphism RIIIB: NA1/NA1, high affin-ity; NA1/NA2, intermediate; and NA2/NA2, low affinity bindingto IgG. We found no association between high and low affinityFcR binding and expansion of CD19.CAR-VSTs (SupplementaryTable S2).

DiscussionWe infused a low dose of second-generation CD19.CAR-VSTs

into 8 patients who had received HSCT for high-risk B-ALL in theabsence of cytoreduction. Although all patients had circulatingnormal CD19þ B cells at the time of infusion, CD19.CAR-VSTexpansion and normal B-cell elimination occurred only in the

Figure 5.

VSTs are not eliminated by monocytes in cocultures. A, CD19.CAR-VSTs or NT-VSTs were cocultured with autologous CD14þmonocytes for 3 days with orwithout EBV pepmixes (px) spanning EBNA1, LMP1, and LMP2 antigens. Viability of CD3þ T cells was assessed on day 3 by flow cytometry with 7ADD (n¼ 3). B,Fold of expansion of CD19.CAR-VSTs and NT-VSTs 3 days after coculture with monocytes with or without EBV pepmix stimulation. Cells were harvested andanalyzed by flow cytometry with Dynabeads to obtain absolute cell numbers (n¼ 3). No statistically significant difference for CAR.VSTs pulsed with EBVpepmixes was observed at early days (day 3) after stimulation. C,Monocytes are eliminated on day 1 of coculture with CD19.CAR-VSTs and nontransduced cells.

Lapteva et al.





presence of concomitant TCR stimulation by viral antigens. Thedegree of CD19.CAR-VST expansion and subsequent B-cell apla-sia correlated with EBV load in peripheral blood, suggesting thatin the absence of the lymphoproliferative milieu resulting fromcytoreductive chemotherapy, in patients with low/no diseaseburden, CAR stimulation alone can be insufficient for effectorcell expansion and that this deficiency can be overcome by theaddition of TCR stimulation.

In most clinical studies, significant expansion and functionof CAR-T cells, requires extensive cytoreduction with cytoxanand fludarabine. Among other effects, these drugs producelymphodepletion, which liberates homeostatic cytokines tosupport CAR-T–cell expansion. In this study HSCT recipientsreceived no further cytoreductive therapy before receiving alow dose (107/m2) of CD19.CAR-VSTs between 74 and158 days posttransplant. Although their immune reconstitutionwas incomplete at this time, T and B lymphocytes circulated withnormal frequencies (946 � 569 lymphocytes/mL; Table 1) versusless than 100 lymphocytes/mL after cytoxan and fludara-bine (25, 26). Hence, the presence of near-normal numbers ofcirculating CD19þ B cells was insufficient to induce substantialCD19.CAR-VST expansion.

In patients with EBV reactivation, CD19.CAR-VST expansionand corresponding elimination of normal B cells occurredwithout cytokine release syndrome (CRS), even when theCD19.CAR-VST cells comprised 32% of all peripheral CD3þ Tcells (30,000 transgene copies/mg DNA in patient 1). CRS hasbeen associated with lymphodepletion and high tumor burden,neither of which applied to our patients. In several clinical trialswe have observed massive expansion of virus-specific T-cells inHSCT recipients with virus infection or reactivation, includingseveral with bulky EBV-driven lymphoma, and none of thesepatients experienced severe CRS or neurotoxicity. It is possiblethat VSTs that are derived from thememory compartment are lessreactive with myeloid cells than na€�ve cells that are expandedpolyclonally in most clinical trials of CAR-T cells. This virusspecificity likely also explains why none of our patients experi-enced GVHD, even during CD19.CAR-VST expansion, anddespite HLA mismatches in 2 patients. This has been a consistentfinding in patients receiving virus-specific T cells from allogeneicdonors (27), even when derived from partially HLA-mismatchedthird-party donors HLA-matched at only one or two alleles (28),and is likely a result of the greatly reduced TCR repertoire of T cellsspecific activated with viral antigens rather than polyclonallyactivated with CD3 and CD28 antibodies."

Although our results suggest that stimulation through the TCRcanpromote the activity ofCD19.CAR-VSTs, repeated stimulationvia the CAR may not always prove beneficial for TCR expres-sion (29, 30). As illustrated in Supplementary Fig. S5, repeatedstimulation in vitro via the CAR can lead to downregulation of theTCR. This may explain the lack of apparent expansion of VSTs inpatient 4. At the time of infusion, this patient had a measurableEBV load that responded rapidly to CD19.CAR-VSTs, whichexpanded equally rapidly as measured by PCR and eliminatednormal B cells. However, there was no apparent increase in thefrequency of circulating T cells for EBV, CMV, or adenovirus asmeasured by IFNg ELISpot assay. In contrast, in patients 1 and 3who had a high virus load, CD19.CAR-VST expansion correlatedwith both a decrease in virus load and an increase in the frequencyof EBVSTs. This paradox is difficult to explain and is the subject offurther studies.

The substantial in vivo expansion and persistence of CD19.CAR-VSTs in the presence of virus reactivation suggestedthat the CH2.CH3 domain of IgG1 does not lead to lungentrapment or activation-induced cell death as has beenobserved in animal models using CARs containing CH2.CH3domains from IgG4 (24). Supporting this suggestion, in vitrococulture of our IgG1 domain-containing CD19.CAR-VSTswith FcR-positive monocytes or NK cells did not result inT-cell death, nor did the affinity of the CD32 and CD16 FcRsin these patients alter the performance of the CAR-VST,whose behavior was unaffected by whether the patientsexpressed the high- or low-affinity polymorphisms of thesereceptors.

In this study, CD19.CAR-VSTs expanded to a greater degree inthe presence of virus reactivation than observed with our earlierstudy using VSTs transduced with the same CD19.CAR (ref. 15;Supplementary Fig. S6). These related studies used differentapproaches to manufacture CD19.CAR-VST. In the earlier study,VSTs were transduced on day 19 of culture, 3 days after the thirdstimulation when T cells demonstrated an effector memoryphenotype. In this study, we transduced VSTs 3 days after thefirst stimulation, when T cells with a central memory phenotypeand long-termmemory potential were transduced (16).Whenweanalyzed CARþ T cells isolated from patient 4, 3 weeksafter infusion we found the proportion of central memory T cellswas higher than in the infused T-cell line (54% vs. 4.3%CD45ROþCCR7þ of CD19.CARþCD3þ T cells in the bloodand in the line, respectively), showing that like unmodifiedVSTs, CARþ VSTs were able to reenter the memory compartment(Supplementary Fig. S7). Furthermore, the early-transducedCD19.CAR-VSTs showed greater expansion in the presence orabsence of virus reactivation than in previous study (Supplemen-tary Fig. S6; ref. 15). Therefore, we propose that earlytransduction, allowing infusion of CD19.CAR-VSTswith a centralmemory phenotype was likely a major contributor to thegreater expansion, persistence, and function of CD19.CAR-VSTsin this study.

Extensive CD19.CAR-VST expansion was observed only inpatients who experienced virus reactivation and B-cell depletionoccurred only in these patients. The only virus detected during thepost-infusion period was EBV, so we cannot determine whetherthe same CD19.CAR-VST expansion would have occurred inassociation with CMV or adenovirus reactivation. While we didobserve an increase in the frequency of adenovirus-specific T cellswithout detectable adenovirus in blood, it is possible that thepatient had a subclinical adenovirus infection,with virus presencein other sites such as in stool or urine. Lack of CD19.CAR-VSTexpansion in the absence of virus reactivation suggests thatsignaling through the CAR on its ownwas not sufficient to inducesubstantial CAR-T–cell expansion or function in vivo, despite theCD28-derived costimulatory endodomain and the presence ofnormal CD19þ B cells. In contrast, stimulation via the TCRachieved this goal, supporting our initial hypothesis that in vivoTCR stimulation could enhance the function of the CAR in T cells.These attributable benefits of dual TCR/CAR stimulation mayresult from more physiologic signals transmitted by the TCR orbecause EBV reactivation is associated with inflammation pro-duced by EBV-derived pathogen-associated molecular patternsthat stimulate pattern recognition receptors of the innate immunesystem producing a more conducive microenvironment for T-cellactivation.






In conclusion,wehavedemonstrated that VSTsmodifiedwith aCD19.CAR containing a long IgG1 spacer and a CD28 endodo-main can expand, persist long term, and produce TCR-mediatedantiviral effects through their TCRs and concomitant anti-CD19þ

B-cell activity through their CARs. These benefits are observedeven in patients who are unsuited to potent cytoreductivedrugs and were not associated with toxicities. The use of CD19.CAR-VSTs, perhaps with a viral vaccine,may be of particular valuepost-remission induction for patients with low disease burdenwho are at high risk of relapse and it would be of interest todiscover whether the enhancement attributable to dual receptorstimulation can be extended to other diseases such as breastcancer, in which patients have dormant disseminated tumor cellsthat may on their own be insufficient to produce stimulationthrough a CAR alone.

Disclosure of Potential Conflicts of InterestI. Diaconu is an employee/paid consultant for Bluebirdbio and ElevateBio.

R.H. Rouce reports receiving commercial research grants from TESSA Pharma-ceuticals, reports receiving other commercial research support and speakersbureau honoraria from Novartis and Gilead/Kite Pharma. G. Dotti reportsreceiving other commercial research support from Cell Medica. C.R. Cruz holdsownership interest (including patents) in and is an advisory board member/unpaid consultant for Mana Therapeutics. C.A. Ramos is an employee/paidconsultant for Novartis and Celgene and reports receiving commercial researchgrants from Tessa Therapeutics. H.E. Heslop is an employee/paid consultant forGilead Biosciences, Novartis, Cytosen, Tessa Therapeutics, and Marker Thera-peutics, reports receiving commercial research grants from Cell Medica andTessa Therapeutics, and holds ownership interest (including patents) in AlloVirandMarker Therapeutics. M.K. Brenner is an employee/paid consultant for andholds ownership interest (including patents) in Tessa Therapeutics, MarkerTherapeutics, Allovir, Allogene, and BluebirdBio. C.M. Rooney is an employee/paid consultant for Tessa Therapeutics, holds ownership interest (includingpatents) in Allovir and Marker Therapeutics, while spouse has ownership

interests in Tessa Therapeutics, Allogene, and BluebirdBio. She is also anadvisory board member/unpaid consultant for CellGenix. No potential con-flicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: H. Liu, G. Dotti, H.E. Heslop, M.K. Brenner,C.M. RooneyDevelopment of methodology: N. Lapteva, I. Diaconu, O. Dakhova,B. Savoldo, G. Dotti, A.P. Gee, C.M. RooneyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Lapteva, M. Gilbert, L.A. Rollins, S. Naik,R.A. Krance, T. Tripic, M. Hiregange, R.H. Rouce, B. Omer, A. Durett,E. Pacheco, C.A. Ramos, A. Orozco, K. Sharpe, M. Gates, D. RaghavanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N. Lapteva, I. Diaconu, M. Al-Sabbagh, H. Liu,A. Durett, M.K. Brenner, C.M. Rooney, M. GatesWriting, review, and/or revision of the manuscript: N. Lapteva, M. Gilbert,I. Diaconu, L.A. Rollins, S. Naik, R.A. Krance, R.H. Rouce, B. Omer, G. Dotti,C.R. Cruz, C.A. Ramos, H.E. Heslop, M.K. Brenner, C.M. RooneyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructingdatabases):M.Gilbert,M.Al-Sabbagh, A.Durett, E. Pacheco,C.M. RooneyStudy supervision: E. Pacheco, C.A. RamosOther (cell product manufacturing): A.P. Gee

AcknowledgmentsThis work was supported by grants from V-Foundation, Alex's Lemonade

Stand Foundation, NIH-NCI P50 CA126752 SPORE in lymphoma and Leu-kemia and lymphoma Society SCOR 7001.

The costs of publicationof 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 December 24, 2018; revised April 29, 2019; accepted September 17,2019; published first October 1, 2019.

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Published OnlineFirst September 26, 2019.Clin Cancer Res Natalia Lapteva, Margaret Gilbert, Iulia Diaconu, et al. Cells

Expressing T−Function of CD19 Chimeric Antigen Receptor T-Cell Receptor Stimulation Enhances the Expansion and

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T-Cell Receptor Stimulation Enhances the ......Nov 08, 2019 · We therefore tested the efﬁcacy of CD19.CAR-VSTs in eight HSCT recipients in remission from high-risk, CD19þ B-cell