Antitransgene Rejection Responses Contribute to
`Attenuated Persistence of Adoptively Transferred
`CD20/CD19-Specific Chimeric Antigen Receptor
`Redirected T Cells in Humans
`
`Michael C. Jensen,1,2 Leslie Popplewell,2 Laurence J. Cooper,2 David DiGiusto,2
`Michael Kalos,1 Julie R. Ostberg,1 Stephen J. Forman1,2
`
`Immunotherapeutic ablation of lymphoma is a conceptually attractive treatment strategy that is the subject of
`intense translational research. Cytotoxic T lymphocytes (CTLs) that are genetically modified to express
`CD19- or CD20-specific, single-chain antibody–derived chimeric antigen receptors (CARs) display HLA-
`independent antigen-specific recognition/killing of lymphoma targets. Here, we describe our initial experi-
`ence in applying CAR-redirected autologous CTL adoptive therapy to patients with recurrent lymphoma.
`Using plasmid vector electrotransfer/drug selection systems, cloned and polyclonal CAR1 CTLs were gen-
`erated from autologous peripheral blood mononuclear cells and expanded in vitro to cell numbers sufficient
`for clinical use. In 2 FDA-authorized trials, patients with recurrent diffuse large cell lymphoma were treated
`with cloned CD81 CTLs expressing a CD20-specific CAR (along with NeoR) after autologous hematopoi-
`etic stem cell transplantation, and patients with refractory follicular lymphoma were treated with polyclonal
`T cell preparations expressing a CD19-specific CAR (along with HyTK, a fusion of hygromycin resistance and
`HSV-1 thymidine kinase suicide genes) and low-dose s.c. recombinant human interleukin-2. A total of 15 in-
`fusions were administered (5 at 108cells/m2, 7 at 109cells/m2, and 3 at 2  109cells/m2) to 4 patients. Overt
`toxicities attributable to CTL administration were not observed; however, detection of transferred CTLs in
`the circulation, as measured by quantitative polymerase chain reaction, was short (24 hours to 7 days), and
`cellular antitransgene immune rejection responses were noted in 2 patients. These studies reveal the primary
`barrier to therapeutic efficacy is limited persistence, and provide the rationale to prospectively define T cell
`populations intrinsically programmed for survival after adoptive transfer and to modulate the immune status
`of recipients to prevent/delay antitransgene rejection responses.
`Biol Blood Marrow Transplant 16: 1245-1256 (2010) Ó 2010 American Society for Blood and Marrow Transplantation
`
`KEY WORDS: Cellular immunotherapy, Adoptive therapy, T lymphocyte, Clinical trial
`
`From the 1Department of Cancer Immunotherapeutics and Tumor
`Immunology; and 2Department of Hematology and Hemato-
`poietic Cell Transplant, Beckman Research Institute, City of
`Hope National Medical Center, Duarte, California.
`Laurence J. Cooper is currently at the Division of Pediatrics, M.D.
`Anderson Cancer Center, Houston, Texas. Michael Kalos is
`currently at the Abramson Family Cancer Research Institute,
`University of Pennsylvania School of Medicine, Philadelphia,
`Pennsylvania.
`Financial disclosure: See Acknowledgments, page 1256.
`Correspondence and reprint requests: Michael C. Jensen, MD,
`Department of Cancer Immunotherapeutics and Tumor Immu-
`nology, Beckman Research Institute, City of Hope National
`Medical Center, 1500 East Duarte Road, Duarte, CA 91010-
`3000 (e-mail: mjensen@coh.org).
`Received January 26, 2010; accepted March 11, 2010
`Ó 2010 American Society for Blood and Marrow Transplantation
`1083-8791/$36.00
`doi:10.1016/j.bbmt.2010.03.014
`
`INTRODUCTION
`
`Although conventional chemotherapy, radiation
`therapy, and antibody therapy can be efficacious in
`treating lymphoma, relapse and progressive disease
`are the major sources of patient morbidity and mortal-
`ity [1,2]. Experimental evidence that
`the cellular
`immune system can eradicate lymphoma provides
`a basis for the development of therapies aimed at
`amplifying antitumor immune responses [3,4]. The
`adoptive transfer of lymphoma-specific T cells is one
`strategy to augment antilymphoma immunity. A sig-
`nificant challenge to executing this strategy is the iso-
`lation of T cells specifically reactive to lymphoma.
`Alternately, the ex vivo derivation of tumor-specific
`T lymphocytes by genetic modification to express
`tumor-targeting chimeric antigen receptors (CARs)
`is a rapidly evolving focus of translational cancer
`
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`Biol Blood Marrow Transplant 16:1245-1256, 2010
`
`immunotherapy [5,6]. Antibody-based CARs are
`HLA-unrestricted and thus can be used in patient pop-
`ulations with target-antigen–positive tumors.
`We have constructed 2 CARs specific for the B cell
`lineage antigens CD20 and CD19 for the purpose of
`targeting lymphomas and leukemias [7,8]. When
`expressed in cytotoxic T lymphocytes (CTLs), these
`CARs redirect effector cells to lyse B-lineage lymph-
`oma targets [7,8]. Here we report our initial clinical
`experience in manufacturing and infusing autologous
`T cells expressing CD20R or CD19R in patients with
`relapsed B cell lymphoma under City of Hope–held
`FDA-authorized trials BB-IND-8513/IRB 98142 and
`BB-IND-11411/IRB 01160, respectively.
`
`MATERIALS AND METHODS
`
`Patients
`
`City of Hope Internal Review Board (IRB) proto-
`cols 98142 and 01160 were activated for patient accrual
`following IRB and Institutional Biological Safety
`Committee approval, FDA authorization (BB-IND-
`8513 and BB-IND-11411, respectively), and National
`Institutes of Health Office of Biotechnology Activities
`registration (9907-330 and 0207-543, respectively). In
`brief, for IRB 98142, patients were eligible if they had
`immunohistopathologically documented CD201 dif-
`fuse large cell lymphoma (DLCL) with a history of
`recurrent or refractory disease and did not have central
`nervous system metastases. After leukapheresis, pa-
`tients began salvage/mobilization chemotherapy,
`then underwent hematopoietic stem cell transplanta-
`tion (HSCT). The first of 3 escalating-dose T cell infu-
`sions was given at 28 days post-HSCT. For IRB
`protocol 01160, patients were eligible if they had path-
`ologically documented follicular lymphoma (FL) with
`evidence of progression after previous rituximab ther-
`apy and did not have central nervous system metastases
`or a history of allogeneic HSCT. These patients were
`enrolled no sooner than 3 weeks after their most recent
`cytotoxic chemotherapy.
`
`Plasmid Vectors
`
`The plasmid expression vectors encoding the
`CD20R chimeric immunoreceptor and the neomycin
`phosphotransferase cDNAs and the CD19R chimeric
`immunoreceptor and the selection-suicide HyTK (a
`fusion of hygromycin resistance and HSV-1 thymidine
`kinase suicide genes) cDNAs have been described
`previously [7,8] (Figure S1A). In brief, the chimeric
`construct consists of VH and VL gene segments of
`the CD20-specific Leu-16 or CD19-specific FMC63
`monoclonal antibodies (mAbs), an IgG hinge-CH2-
`CH3 region, a CD4 transmembrane region, and the
`cytoplasmic domain of the CD3z chain (Figure S1B).
`
`Isolation, Transfection, Selection, Cloning, and
`Expansion of T Cells
`
`The methods for OKT3 stimulation of peripheral
`blood mononuclear cells (PBMCs), and for PBMC
`electroporation, selection, cloning (IRB 98142 only),
`and subsequent growth using the rapid expansion
`method (REM), consisting of recursive 14-day cycles
`of activation with OKT3, recombinant human inter-
`leukin (rHuIL)-2, and PBMC/lymphoblastoid cell
`line (LCL)-irradiated feeders, have been described
`previously [9]. The overall T cell product manufactur-
`ing schemas for each trial are depicted in Figure S1C.
`
`Cell Product Quality Control Tests
`
`The cell product quality control tests (QCTs) per-
`formed and the requisite test results for product release
`are summarized in Table S1.
`
`Confirmation of Plasmid Vector Integration (IRB
`98142 Only)
`
`A single site of plasmid vector chromosomal inte-
`gration was confirmed by Southern blot analysis of
`XbaI/HindIII-digested T cell genomic DNA using
`a 420-bp NeoR-specific probe generated using the
`pcDNA3.1(-) plasmid as a template [9]. The pass crite-
`rion of this test was defined as detection of a single
`band.
`
`Confirmation of CAR Expression
`
`Western blot analysis for CAR expression has been
`described previously [10]. In brief, reduced whole-cell
`lysates are subjected to Western blot analysis with an
`anti-human CD3-z (cytoplasmic tail)-specific mAb
`8D3 (BD Pharmingen, San Diego, CA). This probe
`detects both the 16-kDa endogenous z and the 66-
`kDa CAR z. Pass criteria were defined as visualization
`of both the 16-kDa and 66-kDa bands. Flow cytometry
`analysis for surface CAR expression was determined
`using a fluorescein isothiocyanate (FITC)-conjugated
`Fc-specific
`antibody
`(Jackson ImmunoResearch,
`West Grove, PA). Pass criteria were defined as unim-
`odal positive staining for Fc compared with the
`FITC-conjugated isotype control (BD Biosciences,
`San Jose, CA).
`
`Surface Phenotype Determination
`
`T cell products were evaluated for cell-surface
`phenotype using standard staining and flow cytometric
`procedures with FITC-conjugated mAbs (BD Biosci-
`ences), followed by analysis on a FACScaliber analyzer
`(BD Biosciences). The pass criterion was $90% posi-
`tive staining for TCR-ab and CD8 (IRB 98142) or
`CD3 (IRB 01160) compared with the isotype control.
`Independent of the QCT guidelines, other correlative
`surface markers included CD4 for IRB 98142 and both
`CD4 and CD8 for IRB 01160.
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`Adoptive Therapy with Redirected T Cells for Lymphoma
`
`1247
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`Assay for Antilymphoma Cytolytic Activity
`Cytolytic activity of CAR1 CTLs against 51Cr-
`labeled human lymphoma Daudi cells was assessed as
`described previously using a 4-hour chromium release
`assay [8]. The pass criterion was $50% specific lysis at
`an effector-to-target ratio of 25:1.
`
`Viability
`
`Viability was determined by standard trypan blue
`dye exclusion. The pass criterion was .90% viability.
`
`Sensitivity to Ganciclovir Ablation (IRB 01160
`Only)
`
`To test for acquired cytocidal sensitivity to ganci-
`clovir (GCV), aliquots of cells were harvested from
`5-day REM cultures, then maintained for 14 days in
`37.5 U/mL rHuIL-2 with or without 1 mM GCV.
`Then the cells were harvested and subjected to viability
`testing. The pass criterion was #25% viability in the
`GCV-treated cultures.
`
`Assay for Antigen/IL-2–Independent Growth
`First, 5  106 cells were washed and plated in anti-
`gen- and IL-2–free culture media at the end of a 14-day
`REM cycle. Parallel cultures of Jurkat T cells (Ameri-
`can Type Culture Collection, Manassas, VA) (IRB
`98142) or T cells cultured in the presence of 37.5 U/
`mL rHuIL-2 (IRB 01160) served as controls for expan-
`sion and viability. For IRB 98142, following an 11-day
`incubation, cultures were harvested, counted using try-
`pan blue, plated into 96-well plates at 6000 viable cells
`per well, and pulsed with 1 mCi of 3H-TdR. DNA was
`harvested following a 4-hour incubation at 37C. For
`IRB 01160, viable cell numbers of 14-day cultures
`were determined by flow cytometry as described
`previously [11]. Release criteria specified that cells
`must exhibit \10% of the Jurkat cpm (IRB 98142) or
`\10% of the IL-21 control cell number (IRB 01160).
`
`Sterility
`
`Sterility tests were performed according to an FDA
`Center for Biologics and Evaluation of Research–man-
`dated schedule. Aliquots of media from the T cell cul-
`tures were plated onto bacterial and fungal growth
`media. Mycoplasma detection was conducted on me-
`dia aliquots using the Gen-Probe Mycoplasma Tissue
`Culture-NI Rapid Detection System (Gen-Probe, San
`Diego, CA), and endotoxin levels were determined by
`enzyme-linked immunosorbent assay. Pass criteria
`were negative bacterial,
`fungal, and mycoplasma
`results, along with an endotoxin level\5 EU/kg recip-
`ient weight.
`
`numbers before being resuspended in 0.9% NaCl
`with 2% human serum albumin in a clinical reinfusion
`bag. T cells were reinfused i.v. over 30 minutes
`through either a central line or an age-appropriate
`sized i.v. catheter inserted into a peripheral vein. The
`infusion bag was mixed gently every 5 minutes during
`the infusion. The intrapatient dose escalation plan is
`shown schematically in Figure S2. In IRB 01160, flu-
`darabine (Flu) was administered after the first T cell
`infusion as a potential nonmyeloablative (NMA)
`immunosuppressive regimen for attenuating possible
`rejection responses against the transferred T cells.
`The guidelines provided in the National Cancer Insti-
`tute’s (NCI) Common Toxicity Criteria, version 2.0
`(https://ctep.ifo.nih.gov/l) were followed for the mon-
`itoring of toxicity and adverse event reporting. Rules
`for dose escalation, de-escalation, and cancellation
`were strictly enforced and resulted in 3 of the 4 treated
`patients deviating from the planned infusion cell dose
`escalation at least once.
`
`In Vivo Persistence of Transferred T Cells
`
`For IRB 98142, PBMCs from heparinized periph-
`eral blood samples were isolated and analyzed for per-
`centage of transfected cells by quantitative polymerase
`chain reaction (qPCR) as described previously [12]
`using primers and probes to quantify CD20R copy
`number (details available on request).
`For IRB 01160, samples were received, processed,
`stored, and analyzed in accordance with current good
`laboratory practice guidelines. A validated qPCR-
`based assay to quantify CD19R plasmid vector DNA
`in samples was developed and performed using an
`MJ Research DNA engine with a Chromo 4 continu-
`ous fluorescence detector qPCR module (Bio-Rad,
`Hercules, CA). Real-time qPCR was performed in
`a 20-mL reaction mixture volume containing 50 ng
`DNA, 10 mL of IQ SYBR Green Supermix (Bio-Rad;
`catalog no. 170-8880), and 0.5 pmol of each primer.
`Quantification of the CD19R transgene sequence in
`DNA isolated from patient PBMCs was evaluated us-
`ing qPCR to amplify a 182-nucleotide fragment that
`spanned the CD4 transmembrane–zeta junction
`within the transgene coding sequence, and a standard
`curve derived by dilution of DNA isolated from a clone
`with a single integration of the CD19R transgene
`(primers, probes, and amplification conditions avail-
`able on request). The qualification studies for this am-
`plification reaction demonstrated no amplification
`from healthy donor-derived PBMCs (n 5 5), whereas
`the transgene sequence could be quantified in
`a PBMC sample if the transgene containing DNA
`composed as little as 0.1% of the total DNA sample.
`
`Adoptive Transfer of T Cells
`
`Analysis of Antitransgene Rejection Responses
`
`Processed and cryopreserved cell banks were
`thawed and expanded in culture to the desired cell
`
`Pretreatment and post-treatment PBMCs (on days
`175, 177, and 150 from UPN006, UPN009, and
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`UPN035, respectively) were first stimulated with irra-
`diated therapeutic T cells (3000 rads) or LCLs with
`and without pcDNA3.1(-) plasmid (8000 rads) plus ir-
`radiated pretreatment PBMCs as feeder cells at a 10:1
`responder-to-stimulator ratio in RPMI 1640 (Irvine
`Scientific, Santa Ana, CA) supplemented with 2 mM
`L-glutamine (Irvine Scientific), 25 mM Hepes (Irvine
`Scientific), 100 U/mL penicillin, 0.1 mg/mL strepto-
`mycin (Irvine Scientific), and 10% heat-inactivated
`human serum. One week later, the same irradiated
`stimulator, as well as irradiated pretreatment PBMC
`feeders (3500 rads), were added at a 1:1:1 responder-
`to-stimulator-to-feeder ratio. This stimulation schema
`was repeated up to 2 more times (once weekly) until suf-
`ficient numbers were obtained for chromium-release
`assays. Cytolytic activity of these stimulated PBMC
`against 51Cr-labeled targets was analyzed as described
`previously using a 4-hour chromium-release assay [8].
`For IRB 01160, cellular antitransgene immune re-
`sponses were evaluated directly ex vivo using a combina-
`tion of T cell receptor (TCR) Vb spectratyping and
`CD107 degranulation assays. For the TCR Vb spectra-
`typing analysis, RNA was
`isolated from PBMC
`collected before
`and after
`infusion using the
`RNAqueous-4 PCR Kit for Isolation of DNA-free
`RNA (Applied Biosystems/Ambion, Austin, TX), and
`cDNA was then synthesized using the iScript cDNA
`Synthesis Kit (Bio-Rad). TCR Vb spectratyping analy-
`sis was performed on cDNA samples essentially as de-
`scribed previously [13] using pools of Vb-specific
`primers. A parallel series of amplifications using
`cDNA generated from pooled healthy donor PBMCs
`was performed as a quality control for amplification of
`each Vb family. Aliquots of the amplification mixes
`were run on sequencing gels, followed by analyses using
`Genemapper v3.7 software (Applied Biosystems). The
`CD107 degranulation/mobilization assay was per-
`formed essentially as described previously [14], using
`patient PBMCs collected before and after infusion as ef-
`fectors and infused T cell product or OKT3-expanded
`preinfusion PBMCs (i.e., autologous T cells) as targets.
`To detect spontaneous degranulation, a control sample
`without target cells was included in every experiment.
`FITC-conjugated anti-CD107a
`and anti-CD107b
`(BD Biosciences) were added directly to the tubes be-
`fore incubation. After 5 hours of coincubation, cells
`were washed twice
`and stained with PE-Cy5-
`conjugated anti-CD8b and PE-conjugated anti-TCR
`Vb23 (Beckman Coulter, Fullerton, CA) for 30 minutes
`at room temperature in the dark, rewashed, and ana-
`lyzed on a FC500 flow cytometer using FCS Express
`v3.0 software (Beckman Coulter), with gating on
`CD8b1 and Vb231 lymphocytes.
`
`Serologic Anti-CAR Immune Response Analysis
`
`Serum was isolated from patient blood samples
`collected in red-top (no additive) tubes using an
`
`established laboratory standard operating procedure
`and a qualified assay to detect CAR-specific serologic
`responses in samples. Samples were allowed to clot
`for 2-1/2 hours at room temperature, then centrifuged
`at 1000  g for 15 minutes at 4C. Serum was col-
`lected, aliquoted, and frozen immediately at 280C.
`Flow cytometry detection of potential serum antibody
`responses against
`the anti-CD19R transgene was
`performed using parental versus CD19R-expressing
`Jurkat cell lines as indicator cell lines. The presence
`of antibodies in patient serum that specifically bound
`to CD19R1 Jurkat cells was evaluated by a subsequent
`incubation with FITC-conjugated AffiniPure F(ab’)2
`fragment goat anti-human IgG (Fcg; Jackson Immu-
`noResearch). The cutoff for a negative response was
`established by defining the 95% one-sided prediction
`interval using a pool of non–CAR-reactive serum
`samples from healthy volunteers.
`
`RESULTS
`
`Patient Characteristics
`
`The patients in these studies either had DLCL
`(BB-IND 8513/IRB 98142) or
`follicular non-
`Hodgkin lymphoma (NHL; BB-IND 11411/IRB
`01160) (Table S2). One of the 4 research participants
`had bulky disease, including sites at the neck, chest,
`lymph nodes, and pelvis, at the time of enrollment.
`Three of the 4 participants had received rituximab
`(chimeric mAb specific for CD20) therapy before the
`first infusion of therapeutic T cells. The average dura-
`tion of time from leukapheresis to first infusion was
`106 days, and was affected by the time required to
`manufacture the T cell product and/or the timing of
`the patient’s recovery from salvage therapy.
`
`Generation of Genetically Modified T Cells
`
`Cell products meeting all quality control release
`tests (Table S1) were successfully generated for 2 of
`the 5 patients enrolled on IRB 98142, and for both pa-
`tients enrolled on IRB 01160. The failure to release
`products for 3 of the patients enrolled on IRB 98142
`stemmed from an inability to isolate T cell clones
`that expressed CD8, expressed endogenous TCR, or
`expanded adequately in vitro. The results of Southern
`blot analysis indicating the desired single-site inser-
`tions of the CD20R transgene within the released
`clones of IRB 98142 are depicted in Figure 1. Western
`blot and cell-surface expression profiles of T cell
`products for both trials are also depicted, confirming
`expression of the CAR protein. These cells were fur-
`ther subjected to flow cytometry analysis for confirma-
`tion of the T cell subset markers CD4, CD8, and either
`TCR-ab or CD3. All of the cell products used in ther-
`apy also exhibited redirected killing of CD191 and
`CD201 human Daudi lymphoma targets in 4-hour
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`Adoptive Therapy with Redirected T Cells for Lymphoma
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`1249
`
`51
`Cr-Release
`
`GCV Sensitivity IL-2 Dependence
`10 6
`
`10 5
`
`10 4
`
`10 3
`
`CPM
`
`10 2
`Jurkat
`
`1A11
`
`Jurkat
`
`6D10
`
`10 5
`
`10 4
`
`10 3
`
`10 2
`
`CPM
`
`10 8
`
`10 7
`10 6
`
`IL2
`
`(-)
`
`IL2
`
`(-)
`
`10 5
`
`10 4
`
`10 9
`10 8
`10 7
`10 6
`10 5
`10 4
`
`Viable Cell #
`
`N.D.
`
`N.D.
`
`10 8
`
`10 7
`
`10 6
`
`IL2
`
`IL2
`+GCV
`
`Viable Cell #
`
`10 5
`
`10 9
`
`10 8
`
`10 7
`
`10 6
`
`IL2
`
`IL2
`+GCV
`
`50:1 25:1 5:1 1:1
`
`50:1 25:1 5:1 1:1
`
`50:1 25:1 5:1 1:1
`
`50:1 25:1 5:1 1:1
`Effector:Target
`
`100
`80
`60
`40
`20
`
`0
`
`100
`80
`60
`40
`20
`0
`
`100
`80
`60
`40
`20
`0
`
`100
`80
`60
`40
`20
`0
`
`% Specific Lysis
`
`Southern Western
`
`Flow Cytometry
`
`66kd
`
`UPN006
`Clone 1A11
`
`UPN009
`Clone 6D10
`
`UPN035
`
`N.D.
`
`UPN037
`
`N.D.
`
`16 kd
`
`anti-Fc
`
`anti-TCR
`
`anti-CD8
`
`anti-CD4
`
`66kd
`
`16 kd
`
`66kd
`
`16 kd
`
`66kd
`
`anti-Fc
`
`anti-TCR
`
`anti-CD8
`
`anti-CD4
`
`anti-Fc
`
`anti-CD3
`
`anti-CD8
`
`anti-CD4
`
`16 kd
`
`anti-Fc
`
`anti-CD3
`
`anti-CD8
`
`anti-CD4
`
`Figure 1. T cell products meet release requirements. Depicted from left to right: Southern blots of T cell genomic DNA using an HyTK-specific probe
`showing existence of single bands as indicated by arrows; Western blots revealing both the 16-kDA endogenous CD3z and the 66-kDA CE7R chimeric z
`bands detected with anti-human CD3z cytoplasmic tail specific antibody; flow cytometry analysis for surface expression of the chimeric receptor using
`anti-Fc antibody, or for the T cell markers CD8, CD4, and TCR or CD3, where isotype control staining is indicated with the open histogram; ability of
`CTL clones to lyse CD191 CD201 Daudi targets was determined in a 4-hour 51Cr release assay; ganciclovir (GCV) sensitivity using a flow cytometry–
`based assay for viable cell numbers after 14 days of culture with either rHuIL-2 or rHuIL-2 1 GCV; assays for IL-2 dependence were performed using
`3H-thymidine incorporation measurements (cpm) of Jurkat T cells versus the indicated T cell clones after 11 days of culture in the absence of rHuIL-2
`(UPN006, UPN009), or using a flow cytometry–based assay for viable cell numbers after the T cell products were cultured in the presence versus the
`absence of rHuIL-2 for 14 days (UPN035 and UPN037). N.D., not done.
`
`chromium release assays. Furthermore, all of the cell
`lines
`retained their dependence on exogenous
`rHuIL-2 for survival and proliferation, and the
`HyTK-expressing lines of IRB 01160 tested positive
`for sensitivity to GCV-mediated ablation.
`
`Treatment Experience
`
`As depicted in Figure 2, the intrapatient dose esca-
`lations were carried out as planned (compare with
`Figure S2), with the exception that the 1010/m2 cell
`dose was never given in IRB 98142 because of protocol
`toxicity dose modification rules
`(UPN006 and
`UPN009). Indeed, because of grade 2 hepatic toxic-
`ities that were noticed with the first infusion dose of
`108/m2 in UPN006, the second infusion was repeated
`at 108/m2, followed by an escalation to 109/m2 for the
`third infusion. Patient UPN009 exhibited a drop in he-
`moglobin after the second infusion that, although clin-
`ically insignificant (from 10.6 to 8.6), represented
`a Common Toxicity Criteria grade change from grade
`1 to grade 2 anemia status, requiring repetition of the
`
`109/m2 dose for the third infusion based on the proto-
`col’s defined rules for dose escalation. In UPN006, the
`second infusion was cancelled and rescheduled because
`of a puncture in the bag, which compromised the in-
`tegrity of the T cell product. In UPN037, the last in-
`fusion was cancelled because of the detection of
`contaminated T cell product. Neither myeloablation
`(MA) followed by HSCT nor Flu resulted in a drop
`in absolute lymphocyte count (ALC) below the normal
`range (Table S3).
`There were no grade 3 or higher adverse events
`with a possible correlation to administration of 108
`T cells per m2. However, examination of the adverse
`events at 109 T cells per m2 revealed one case of
`grade 3 self-limited lymphopenia in both IRB
`98142 and IRB 01160, possibly attributed to cell
`administration (Table 1). At 2  109 T cells per
`m2, grade 3 lymphopenia and grade 3 eosinophilia
`each occurred once in IRB 01160; both resolved
`spontaneously with no adverse sequelae to the
`patients. Overall, the safety profile of this adoptive
`transfer therapy was acceptable.
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`M. C. Jensen et al.
`
`Biol Blood Marrow Transplant 16:1245-1256, 2010
`
`UPN006
`
`e m C e l l s
`
`S t
`
`BCNU
`
`Cytoxan
`
`VP-16
`
`2
`
`/ m
`
`8
`
`0
`
`s i o
`
`I n f u
`
`n C a
`
`d
`
`2
`
`e l l e
`
`c
`
`n
`
`e #
`
`s
`
`D o
`
`2
`
`/ m
`
`8
`
`0
`
`: 1
`
`s
`
`D o
`
`: 1
`
`3
`
`e #
`
`2
`
`/ m
`
`9
`
`0
`
`: 1
`
`1
`
`e #
`
`s
`
`D o
`
`d -37 d0 d14 d28 d42
`
`e m C e l l s
`
`S t
`
`2
`
`/ m
`
`8
`
`0
`
`s
`
`D o
`
`: 1
`
`2
`
`e #
`
`2
`
`/ m
`
`9
`
`0
`
`2
`
`/ m
`
`9
`
`0
`
`s
`
`D o
`
`: 1
`
`3
`
`e #
`
`: 1
`
`1
`
`e #
`
`s
`
`D o
`
`UPN009
`
`TBI
`
`Cytoxan
`
`VP-16
`
`d -28 d0 d14 d28
`
`UPN035
`
`2
`
`/ m
`
`8
`
`2
`
`9
`
`2
`
`9
`
`2
`
`/ m
`
`9
`
`0
`
`2
`
`/ m
`
`9
`
`0
`
`0
`
`: 1
`
`1
`
`e #
`
`s
`
`D o
`
`0
`
`: 1
`
`2
`
`0
`
`: 1
`
`3
`
`/ m
`
`e #
`
`s
`
`D o
`
`/ m
`
`e #
`
`s
`
`D o
`
`1
`
`x
`
`: 2
`
`4
`
`e #
`
`s
`
`D o
`
`1
`
`x
`
`: 2
`
`5
`
`e #
`
`s
`
`D o
`
`d0 d4 d8 d14 d21 d26 d28 d29 d31 d35
`
`Fludarabine
`
`IL-2
`
`IL-2
`
`2
`
`/ m
`
`8
`
`0
`
`UPN037
`
`: 1
`
`1
`
`e #
`
`s
`
`D o
`
`2
`
`/ m
`
`9
`
`0
`
`2
`
`/ m
`
`9
`
`0
`
`s
`
`D o
`
`: 1
`
`3
`
`e #
`
`: 1
`
`2
`
`e #
`
`s
`
`D o
`
`2
`
`/ m
`
`9
`
`0
`
`1
`
`x
`
`s i o
`
`I n f u
`
`: 2
`
`4
`
`e #
`
`s
`
`D o
`
`d
`
`e l l e
`
`c
`
`n
`
`n C a
`
`d0 d4 d8 d14 d21 d22 d26 d28 d30 d35 d38
`
`Fludarabine
`
`IL-2
`
`IL-2
`
`Figure 2. Treatment regimens for each patient. First i.v. infusions of T cells were administered on day 0 for each patient. For UPN006 and UPN009,
`fractionated total body irradiation (TBI) and/or myeloablative chemotherapies administered to UPN006 and UPN009 are indicated just before admin-
`istration of CD341 autologous stem cells. BCNU, bis-chloronitrosourea; cytoxan, cyclophosphamide; VP-16, etoposide. For UPN035 and UPN037,
`administration of fludarabine (i.v. at 25 mg/m2) occurred between days 4 and 8 after the first T cell infusion, and rHuIL-2 administration (5  105
`IU/ m2 BID) was initiated after the third T cell infusion.
`
`Follow-Up Clinical Status of Patients
`
`Although this was a phase I clinical trial with a pri-
`mary purpose of determining safety, we also moni-
`tored the disease and survival status of each patient.
`For IRB 98142 (CD20R; DLCL), UPN006 (last infu-
`sion on February 24, 2000) relapsed in September
`2001, whereas UPN009 (last infusion on November
`22, 2000) continues to be in remission after autologous
`HSCT. At the time of the writing of this report, both
`UPN006 (after additional treatment) and UPN009 are
`alive and in remission. For IRB 01160 (CD19R; FL),
`UPN035 (last infusion on May 4, 2006) presented
`
`with a new diagnosis of CD191/CD201 DLCL in
`June 2006, and died in June 2007. UPN037 (last infu-
`sion on February 15, 2007) displayed progression on
`computed tomography scan in September 2007, and
`is currently alive and undergoing additional treatment.
`
`In Vivo Persistence of Transferred T Cells
`
`Quantitative PCR performed to detect CD20R
`and CD19R plasmid copy numbers in PBMCs as a sur-
`rogate marker of the presence of adoptively transferred
`T cells showed varying T cell persistence among pa-
`tients (Figure 3). Only 1 of the 4 patients (UPN006)
`
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`Biol Blood Marrow Transplant 16:1245-1256, 2010
`
`Adoptive Therapy with Redirected T Cells for Lymphoma
`
`1251
`
`A
`
`UPN
`006
`
`UPN
`009
`
`B
`
`UPN
`035
`
`CD19R+ cells in PBMC
`
`UPN
`037 %
`
`109/m2
`
`108/m2
`
`108/m2
`
`0.015
`
`0.010
`
`* *
`
`109/m2
`
`109/m2
`
`108/m2
`
`* * *
`
`56
`49
`43
`42
`35
`29
`28
`27
`25
`15
`14
`
`017
`
`Day
`
`0.005
`
`0
`0.025
`
`0.020
`
`0.015
`
`0.010
`
`0.005
`
`0
`
`%CD20R+ cells in PBMC
`
`*
`
`2x109/m2
`
`2x109/m2
`
`*
`
`2x109/m2
`
`109/m2
`
`109/m2
`
`108/m2
`
`0.100
`
`0.075
`
`109/m2
`
`109/m2
`
`108/m2
`
`0.050
`
`0.025
`
`0
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0
`
`* *
`
`42
`41
`36
`35
`29
`28
`26
`22
`21
`15
`14
`
`Day
`
`014
`
`Figure 3. Transferred T cells do not persist long term in vivo. Using
`real time quantitative PCR, the percent of cells in the PBMC that
`were positive for the CD20R (A) or CD19R (B) genes were determined
`as an indicator of the relative amount of chimeric receptor expressing T
`cells in the PBMC samples collected at the indicated days during the
`treatment schedule. Escalating infusion doses are indicated by arrows.
`*Cells not harvested.
`
`irradiated 6D10 cells were cloned in limiting dilution;
`all clones were similarly specific for NeoR (Figure 4C).
`For IRB 01160 (CD19R; FL), the development of
`both antibody and cellular immune responses against
`the infused T cell products was evaluated. For the an-
`tibody analyses, serum collected from UPN035 and
`UPN037 at enrollment and after T cell administration
`were both negative for antibody reactivity against the
`surface-expressed CD19R transgene using a flow
`cytometry–based assay (Figure 5A); however, evidence
`was found for cell-mediated immunoreactivity against
`the infused T cells. Examination of the TCR Vb gene
`
`Table 1. Adverse Event Summary
`
`IRB Trial
`
`98142
`
`01160
`
`T Cell
`Dose, Cells/m2
`
`109
`
`109
`2  109
`
`Event*
`
`Occurrences
`
`Lymphopenia
`
`Lymphopenia
`Lymphopenia
`Eosinophilia
`
`1
`
`1
`1
`1
`
`IRB indicates internal review board; NCI, National Cancer Institute.
`*Only events of grade 3 or higher, according to the NCI Common Toxicity
`Criteria, with possible attribution to T cell administration are reported.
`
`had a detectable level of transferred T cells at 1 week
`after the first infusion of 108 cells/m2. A detectable
`level of transferred T cells at 1 week after infusion of
`109 cells/m2 was found in only 2 of the 7 higher doses
`(UPN006 infusion 3 and UPN009 infusion 2), and at
`no time were transferred T cells detected at 1 week af-
`ter infusion of 2  109 cells/m2. Thus, adoptively
`transferred T cell persistence did not appear to corre-
`late with cell dose. Compared with the persistence af-
`ter the initial
`infusion, UPN009, UPN035, and
`UPN037 also displayed significantly reduced levels
`of transferred T cells 24 hours after each additional in-
`fusion, suggesting the possibility of an antitransgene
`immune response mounted against the administered
`T cells (Figure 3).
`
`Detection of Transgene-Specific Immune
`Responses
`
`For IRB 98142 (CD20R; DLCL), the develop-
`ment of cellular immune responses against the infused
`T cell products was evaluated. For these analyses,
`PBMCs were collected from UPN006 and UPN009
`before and after T cell administration, and, after in vi-
`tro stimulation, were compared for cytotoxic activity
`using chromium-release assays (Figure 4). The use of
`irradiated lymphoblastoid cells (xLCLs) to stimulate
`the PBMCs resulted in successful lysis of 51Cr-labeled
`LCLs, indicating that functional effector cells could be
`derived from each patient’s PBMC samples. Interest-
`ingly, when the irradiated autologous T cell clones
`were used to stimulate the PBMCs, cytotoxic re-
`sponses were seen against the 51Cr-labeled T cell clone
`only in the posttreatment sample collected from
`UPN009 (Figure 4A). This immunoreactivity against
`the T cell clone 6D10 used in therapy appeared to be
`specific for neomycin phosphotransferase but not the
`CAR, because cytotoxic responses could be observed
`against 51Cr-labeled LCLs that had been transduced
`with the pcDNA3.1(-) vector, which directs the ex-
`pression of neomycin phosphotransferase, but lacks
`the CD20R transgene (Figure 4B). To better analyze
`the specificity of the rejection response, UPN009’s
`posttreatment PBMCs that had been stimulated with
`
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`

`

`1252
`
`M. C. Jensen et al.
`
`Biol Blood Marrow Transplant 16:1245-1256, 2010
`
`Pre-Trtmt
`
`Post-Trtmt
`
`50:1
`
`25:1
`
`5:1
`
`1:1
`50:1
`Effector:Target
`
`25:1
`
`5:1
`
`1:1
`
`Pre-Trtmt
`
`Post-Trtmt
`
`100
`80
`
`% Cr Release
`
`51
`
`60
`40
`20
`0
`
`50:1
`
`25:1
`
`5:1
`
`1:1
`50:1
`Effector:Target
`
`25:1
`
`5:1
`
`1:1
`
`A
`
`UPN
`006
`
`UPN
`009
`
`B
`
`UPN
`009
`
`100
`80
`60
`40
`20
`0
`
`100
`80
`
`60
`40
`20
`0
`
`% Cr Release
`
`51
`
`Stim/Target:
`
`xLCL/LCL
`xLCL/1A11
`x1A11/LCL
`x1A11/1A11
`
`Stim/Target:
`
`xLCL/LCL
`xLCL/6D10
`x6D10/LCL
`x6D10/6D10
`
`Stim/Target:
`
`xLCL/LCL
`xLCL/LCLpcDNA
`x6D10/LCL
`x6D10/LCLpcDNA
`
`Target:
`
`LCL
`6D10
`LCLpcDNA
`
`F2
`
`E2
`
`D7
`
`D4
`
`Post-Trtmt PBMC clones
`
`(stim w/ x6D10)
`
`C
`
`UPN
`009
`
`-25 0 25 50 75 100 125 150 175 200
`51
`% Cr Release (25:1)
`
`Figure 4. Transgene rejection response detected when T cells administered after HSCT. In the trial targeting CD201 diffuse large-cell lymphoma,
`PBMCs collected before treatment